Chimaeric gene coding for a transit peptide and a heterologous polypeptide

ABSTRACT

Chimaeric DNA sequence which encodes: 1) a transmit peptide of a cytoplasmic precursor of a chloroplast protein or polypeptide of a plant and 2) a protein or polypeptide that is heterologous to the transit peptide. The chimaeric DNA sequence can be used as a vector for transforming a plant cell so that a chimaeric precursor of the heterologous protein or polypeptide is produced in the cytoplasm of the cell and the chimaeric precursor then transports the heterologous protein or polypeptide in vivo into a chloroplast of the cell.

This application is a divisional of application Ser. No. 08/430,257filed Apr. 28, 1995, now U.S. Pat. No. 5,728,925, which was acontinuation of application Ser. No. 08/267,306 filed Jun. 29, 1994, nowabandoned, which was a continuation of application Ser. No. 08/026,213filed Mar. 1, 1993, now abandoned, which was a continuation ofapplication Ser. No. 07/794,635 filed Nov. 18, 1991, now abandoned,which was a continuation of application Ser. No. 07/480,343 filed Feb.14, 1990, now abandoned, which was a continuation of application Ser.No. 06/755,173 filed Jul. 15, 1985, now abandoned, the entire contentsof which are hereby incorporated by reference.

FIELD OF THE INVENTION

The invention relates to means, particular recombinant vectors, and toprocesses for the controlled introduction of foreign gene products inplant chloroplasts.

The disclosure which follows contains reference numbers in the form ofexponents. They refer to bibliographic references relative to literaturereferred to at the end of this specification. Other literature is alsoreferred to in the course of this description by the name of the firstauthor and date of publications. All the articles as well as the patentapplications, patents, etc., which shall be referred to throughout thisspecification are incorporated herein by reference.

It is well known that the cells of eukaryotic organisms, and moreparticularly plant cells, contain distinct subcellular compartments, ororganelles, delimited by characteristic membrane systems and performingspecialized functions within the cell. In photosynthetic leaf cells ofhigher plants the most conspicuous organelles are the chloroplasts,which exist in a semi-autonomous fashion within the cell, containingtheir own genetic system and protein synthesis machinery, but relyingupon a close cooperation with the nucleo-cytoplasmic system in theirdevelopment and biosynthetic activities¹.

The most essential function of chloroplasts is the performance of thelight-driven reactions of photosynthesis. But chloroplasts also carryout many other biosynthetic processes of importance to the plant cell.For example, all of the cell's fatty acids are made by enzymes locatedin the chloroplast stroma, using the ATP, NAOPH, and carbohydratesreadily available there. Moreover, the reducing power of light activatedelectrons drives the reduction of nitrite NO⁻⁻ ₂) to ammonia (NH₃) inthe chloroplast; this ammonia provides the plant with nitrogen requiredfor the synthesis of amino acids and nucleotides.

The chloroplast also takes part in processes of particular concern tothe agrochemical industry.

Particularly it is known that many herbicides act by blocking functionswhich are performed within the chloroplast. Recent studies haveidentified the specific target of several herbicides. For instance,triazine derived herbicides inhibit photosynthesis by displacing aplastoquinone molecule from its binding site in the 32 Kd polypeptide ofthe photosystem II. This 32 Kd polypeptide is encoded in the chloroplastgenome and synthesized by the organelle machinery. Mutant plants havebeen obtained which are resistant to triazine herbicides. These plantscontain a mutant 32 Kd protein from which the plastoquinone can nolonger be displace by triazine herbicides.

Several other herbicides are known to block specific steps in aminoacids synthesis. Sulfonyl-uras are known to inhibit acetolactatesynthase. This enzyme is involved in isoleucine and valine synthesis.Glyphosate inhibits the function of 5-enol pyruvyl-3-phosphoshikimatesynthase, which is an enzyme involved in the synthesis of aromatic aminoacids. All these enzymes are encoded by the nuclear genome, but they aretranslocated into the chloroplast where the actual amino acids synthesistakes place.

Enzymes responsible for the same functions are also present inprokaryotes. It should be easy to obtain bacterial mutants in which theenzyme of interest is no longer sensitive to the herbicide. Such astrategy was used with success to isolate Salmonella typhimurium mutantswith an altered aro A gene product, which confers resistance toglyphosate (Comai et al Science 221, 370 (1983).

Thus the use of chloroplastic or bacterial genes to confer herbicideresistance to plant cells could be successful if their gene productswere efficiently transported into the chloroplast where they function.

Chloroplasts are also involved in the complex mechanisms which regulatethe levels of amino acids synthesis. One of the most importantregulatory mechanisms is the so-called retroregulation. This mechanisminvolves the inhibition of the key enzyme of a given pathway by the endproduct(s) of this pathway. When a key enzyme is no longer subjected tosuch regulation the organism overproduces the corresponding end product(e.g., an amino acid).

Isolation of mutant genes encoding for enzymes that are insensitive toinhibition by the corresponding end product is well documented inbacteria. Similar mutants in plant cells are difficult to obtain andonly a few examples have been reported. Furthermore the isolation ofgenes from plant cells is a very complex task when compared to theisolation of bacterial genes.

As mentioned earlier, most amino acids synthesis take place inside thechloroplast.

Thus, there is a great interest for the development of a technique fortransforming plant cells with bacterial genes encoding an enzymeinsensitive to inhibition by the abovesaid end product in a way suchthat the result of this transformation process would be the introductionof said enzyme in the plant chloroplasts. The ultimate result of thisprocess would be an over-production of amino acid.

These are but a few examples (additional examples will be mentionedlater) of the prospects of considerable development of plant geneticengineering which will be at hand for the specialists as soon aspractical techniques suitable for the introduction of determined foreignpolypeptides or proteins in chloroplasts become available.

Indeed, many techniques have been proposed for the transfer of DNA toplants such as direct DNA uptake, micro-injection of pure DNA and theuse of viral or plasmid vectors. Plasmid vectors which have provenparticularly efficient are those derived from tumor-inducing (Ti)plasmids of the microorganism Agrobacterium tumefaciens which is theagent of crown gall disease in dicotyledonous plants. Those plasmids canbe modified by removal of the tumor-causing genes from their T-DNA. Theso modified plasmids then no longer interfere with normal plant growthand differentiation and, upon insertion of a determined foreign gene atan appropriate site of said plasmids, can be used for promoting theexpression of the protein encoded by said determined gene in plantcells. Particularly, the foreign gene may be inserted close to one ofthe border sequences or between the two border sequences which surroundthe T-DNA in intact Ti-plasmids. Reference can be made by way ofexamples to the articles of:

A. CAPLAN et al. titled "Introduction of Genetic Material into PlantCells", Science, Nov. 18, 1983, volume 222, pp. 815-812;

O. HERRERA-ESTRELLA et al. titled "Expression of chimaeric genestransferred into plant genes using a Ti-plasmid-derived vectors",Nature, Vol. 303, No. 5914, pp. 209-213, May 13, 1983;

L. HERRERA-ESTRELLA et al. titled "Light-inducible and chloroplastassociated expression of a chimaeric gene introduced into Nicotianatabacum using a Ti-plasmid vector", Nature, vol. 310, n⁺ 5973, pp.115-120, Jul. 12, 1984;

or to European Patent Application N⁺ 0 116 718 (or to U.S. applicationSer. No. 570,646) or to the International Application WO 84/02913published under the PCT;

all of these articles or patent applications being incorporated hereinby reference.

Yet all of these techniques do not provide an efficient and relativelyeasy method of transformation of chloroplasts, despite the considerablework which has been devoted to the subject and the already large amountof knowledge which has been acquired, particularly concerning theproduction of proteins in plant cells and transfer into thechloroplasts. As a matter of fact vectors for the direct transformationof chloroplasts are unavailable at this time. Furthermore matureproteins, including those normally encoded in the plant cells includingsaid chloroplasts and which can ultimately be isolated naturally fromsaid chloroplasts, cannot as such be caused to penetrate in thechloroplasts if supplied thereto from outside.

Most chloroplast proteins are coded for in the nuclear DNA and are theproducts of protein synthesis on cytoplasmic ribosomes, many as solublehigher molecular weight precursors²⁻⁹. These precursors are thentranslocated through either one or both of the plastid envelopemembranes, processed, and assembled into their final organellarcompartment or holoenzyme complex. In vitro reconstitution experimentsusing isolated chloroplasts, have demonstrated that the uptake andprocessing of over one hundred nuclear-encoded, cytoplasmicallysynthesized precursors by chloroplasts occurs by an energy-dependent¹⁷,post-translational mechanism ⁶, ¹⁰⁻¹⁷.

The most extensively characterized of these nuclear-encoded chloroplastproteins is the small subunit of ribulose-1,5-bisphosphate (RuBP)carboxylase. This polypeptide is synthesized on free cytoplasmicribosomes as a precursor of 20,000 daltons containing an amino terminalextension or transit peptide of approximately 5-6,000 daltons⁶⁻⁷, ⁹.During or immediately after import of the precursor into thechloroplast, the transit peptide is proteolytically removed in two stepsby a soluble protease¹⁸, yielding a mature small subunit polypeptide of15,000 daltons. This polypeptide is then assembled with an endogenouslarge subunit into the functional RuBP carboxylase holoenzyme¹¹,12.

Similar observations were made with the chlorophyl a/b binding proteins.These polypeptides are synthesized as soluble precursors on cytoplasmidribosomes (Apel and Kloppstech, 1978; Schmidt et al., 1981) and arepost-translationally translocated into chloroplasts. During or aftertranslocation the NH₂ -terminal transit peptides are proteolyticallycleaved (Schmidt et al., 1981) to yield the mature polypeptides. Themature A and B polypeptides associated with chlorophyl a and b areintegrated into the thylacoid membrane. The transit peptides ofpost-translationally transported chloroplast proteins are characterizedby a preponderance of basic amino acids, a feature which has beenproposed as important in the interaction of the transit peptide with thechloroplast envelope¹⁹. Comparison of transit peptides of small sub-unitprecursors from various plant species show a variation in amino acidsequence, but a relatively strong conservation in the position ofprolines and charged amino acid residues ²⁰⁻²² and a substantialhomology in a region surrounding the cleavage site of the precursors, asobserved in soybean (Berry-Lowe et al.; 1982) pea (Cashmore, 1983), duckweed (Stiekema et al., 1983) and wheat (Broglie et al.; 1983). Thesecommon properties may be of functional significance since both invitro¹¹,12 and in vivo²³, the small subunit precursors from one plantspecies can be imported and correctly processes by the chloroplasts ofothers and vice-versa.

The molecular basis of how the post-translational translocation ofpolypeptides into chloroplasts occurs and which signals are involved inthis process, more particularly the relative contributions of thetransit peptide and the mature protein to the uptake and processingmechanism are still not fully understood, even though it was alreadypresumed that the transit peptide is required for the translocation ofthe mature protein. Consistent with this is the observation that themature small subunit protein is not translocated into chloroplasts²⁴.

The invention stems from several discoveries which have been made byApplicants, as a result of further studies of the translocationmechanisms through the chloroplast membranes of chloroplast-proteinprecursors encoded by the nuclear DNA of plant cells. It seems that notcytoplasmic factor is required for the translocation mechanism itself asa result of further studies carried out on RuBP.

Further it has been found that all the sequence information required fortranslocation and transport of the mature protein or of a subunitthereof through the chloroplast membranes seems to reside within theprecursor subunits and even within the sole transit peptides normallyassociated therewith. It further appeared that transit peptides not onlymediate translocation, but also include information necessary forsite-specific processing of the corresponding proteins.

These different properties of the transit peptides are at the basis ofthe recombinant DNAs, more particularly recombinant vectors including aDNA sequence coding for a determined protein or polypeptide,particularly a foreign protein, sought to be introduced and processed inchloroplasts, as well as the processes for the introduction of suchforeign polypeptide or protein into the chloroplasts, for instance inthe thylacoid membranes or, preferably, in the stroma thereof. As amatter of fact an essential element of these recombinant vectorsconsists of a DNA sequence coding for a transit peptide, it beingunderstood that this expression, as used throughout this specificationand claims attached thereto, designates the amino acid sequencecontained in any chloroplast protein precursor which, upon import of theprecursor, is proteolytically removed during or immediately after importof the precursor into the chloroplast to yield either the correspondingfunctional mature protein or a subunit thereof, particularly when, likein the case of RuBP, the final processing of the mature protein takesplace within the chloroplast. Such final processing comprises, forinstance, the assembling of said subunit with another endogenous subunitto yield the final functional protein.

SUMMARY OF THE INVENTION

The recombinant DNA according to the invention which can be introducedinto plant cells is characterized by the presence therein of a chimaericgene comprising a first nucleic acid and a second nucleic acidrecombined with each other, said first nucleic acid and said secondnucleic acid being of different origins, particularly being foreign toeach other, wherein said first nucleic acid contains a first codingsequence which has essential sequence homology with a natural genesequence coding for a transit peptide belonging to a precursorcomprising at least the N-terminal sub-unit of a chloroplast proteincapable of being transported into or processed within the chloroplast ofsaid plant cells and wherein said second nucleic acid contains a secondcoding sequence-distinct of the gene sequence coding for saidchloroplast protein or chloroplast protein sub-unit, said second nucleicacid being located downstream of said first nucleic acid in thedirection of the transcription of their first and second sequencesrespectively.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A diagrammatically represents the successive steps of theconstruction of preferred recombinant DNAs, including a vector suitablefor the transformation of plant cells, containing a chimaeric geneaccording to a first preferred embodiment of this invention;

FIG. 1B represents the structure of the characteristic portion of thechimaeric gene according to this invention and included in the abovesaidrecombinant DNAs;

FIG. 2 shows the results obtained in Southern hybridization experimentswith recombinant DNAs according to the invention, in relation to thedetection of the incorporation of the abovesaid chimaeric gene in thegenome of plant cells;

FIG. 3 is a schematic representation of the organization of the genefusion and the plant-vector sequences of the vector of FIG. 1A, asmodified by said chimaeric gene;

FIG. 4 shows the results obtained in RNA-hybridization experimentscarried out in relation to the detection of the transcriptional activityof the promoter included in the chimaeric gene of the invention;

FIGS. 5A and 5B show comparative results of transcription experimentsunder the control of a light-dependent promoter in plant materialstransformed by the recombinant DNAs of the invention;

FIG. 6A is representative of the results obtained in experimentspurporting to demonstrate the transport of the products encoded by theabove said chimaeric gene into the chloroplasts of plant cells;

FIG. 6B is a graphic display of the relative mobility of the differentactivities of the gene-produce detected in FIG. 6A;

FIG. 7 illustrates results obtained in assays (to show thelight-dependent expression of the fusion protein encoded by the chimaricgene);

FIG. 8A diagrammatically represents successive steps of the constructionof preferred recombinant DNAs according to a second preferred embodimentof the invention, as well as of other recombinant DNAs for studypurposes;

FIG. 8B represents the amino acid sequences encoded by a portion of achimaeric gene diagrammatically shown in FIG. 8A particularly at thejunction of the DNA sequence coding for the selected transit peptide ofa gene encoding the amino terminus of the bacterial neomycinephosphotransferate II (NPT(II) used as a model of protein of bacterialorigin transportable into the chloroplasts;

FIG. 9 shows the results of Southern hybridization analysis ofAgrobacterium and plant DNA as described in Example II;

FIG. 10 is an autoradiogram showing the localization of NPT(II) activityin chloroplasts of tobacco callus tissue as described in Example II;

FIG. 11 is an autoradiogram showing the protection of the NPT(II)activity present within chloroplasts of pGV3851:pGSSTneo3-transformedtobacco cells to protease treatment as described in Example II;

FIG. 12 is an autoradiogram showing the localization of NPT(II) activityin the stromal fraction of chloroplasts isolated frompGV3851:pGSSTneo3-transformed tobacco tissue as described in Example II;

FIG. 13 is an autoradiogram showing the in vitro uptake of TP-NPT(II)fusion protein by isolated pea chloroplasts as described in Example II;

FIG. 14 shows the construction of a plasmid containing a chimeric geneencoding the TP-NPT(II) fusion protein and wherein the coding sequencesare under control of a foreign promoter.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In joint efforts of the inventors to solve the problem sought, i.e.,providing methods and means for transporting a protein intochloroplasts, the inventors have devised two main approaches which havein common the use of recombinant DNAs including a sequence coding for atransit peptide. These two approaches resulted in the two preferredembodiments which will be exemplified hereafter and which both proved tobe effective.

Example I is illustrative of the first approach which took into accountthe possibility that a larger part of the nuclear genes, including theentire region of high homology around the cleavage site of theprecursors would be a necessary requirement for transport and processingof proteins, particularly foreign proteins, into chloroplasts. Thisresulted in a first series of preferred recombinant DNAs of thisinvention more particularly characterized in that the first nucleic acidas defined hereabove contains a third sequence corresponding to at leastpart of a nucleic acid encoding the N-terminal cytoplasmic subunit of achloroplast protein downstream of said first sequence and in that theextremity of said third sequence is substantially contiguous to theextremity of said first nucleic acid.

Preferably the third sequence does not extend beyond the nucleotidesencoding the N-terminal extremity of the cytoplasmic subunit of saidchloroplast protein, yet it comprises an intron, particularly that whichinitially belonged to the same gene as the exons encoding the peptidicportions which will ultimately provide the precursor subunit of thecorresponding natural chloroplast protein.

It will be seen that a preferred embodiment of a recombinant DNAcorresponding to the abovesaid first approach includes a first sequenceencoding the transit peptide and a third sequence which initiallybelonged to the same gene and which encodes the first 22 amino acids ofthe small subunit gene (rbcS) from Pisium sativum (Cashmore, 1983), saidthird gene being then fused to the coding region of a foreign protein,such as the nptII gene which codes for neomycine phosphotransferase II(npt(II) gene obtained from a Tn5 transposon).

Example II is illustrative of the construction which can be made upontaking the second approach to solve the same problems. In thisconstruction, the nptII coding sequence is fused directly to the transitpeptide coding sequence such that the potential protein cleavage sitedoes not contain any amino acids derived from the mature small subunitprotein except the methionine following the last amino acid of thetransit peptide. More generally, and preferably, the first codon of thesecond sequence (encoding the protein, particularly a foreign protein,which is sought to be translocated into the chloroplasts) is immediatelyadjacent to the last codon of said first DNA sequence coding for saidtransit peptide. Thus, particularly when the abovesaid second sequenceencodes a polypeptide or protein different from the chloroplast proteinnormally associated with the transit peptide encoded by said firstsequence, the nucleotide sequence next to said first sequence in saidchimaeric gene is generally (possibly except for a first codon codingfor methionine) by free of sequence homology with the nucleotidesequence encoding the N-terminal part of the normal chloroplast protein.Yet, the last codon of the first sequence and the first codon of thesecond sequence may be separated by any number of nucleotide triplets,preferably in the absence of any intron of stop codon. For instance, a"third sequence" encoding the first amino acids of the mature proteinnormally associated with the transit peptide concerned in thecorresponding natural precursor (particularly those encoded by the exoncontaining the first sequence encoding said transit peptide) may bepresent between said first and second sequences. This "third sequence"may consist of the region of high homology which the N-terminal parts ofcytoplasmic precursor-subunits of chloroplast proteins from soybean,pea, duck-weed and wheat have in common. For instance such "thirdsequence" (be it in the constructions resulting from the "firstapproach" or those from the "second approach" considered hereabove)encodes the pentapeptide sequence M-Q-V-W-P. These letters correspond tothe standard one-letter-abbreviated designations of the natural aminoacids. Obviously other "third seqeuences" of nucleotide-sequences can becontemplated upstream or/and also downstream of the above-defined secondsequence to the extend where the amino acid sequences encoded are notlikely to alter significantly the biological properties of the hybridprotein then formed and translocated into the chloroplasts. Yet in mostpreferred constructions according to that type the second sequence ispreferably fused in phase-register with the first sequence directlycontiguous thereto as mentioned earlier or separated therefrom by nomore than a short peptide sequence, such as that encoded by a syntheticnucleotide-linker possibly used for achieving the fusion. As shown byExample II such a construction is then capable of ensuring thetranslocation into the chloroplasts of any protein or protein fragmentof controlled amino acid sequence, for instance a bacterial protein orprotein fragment or a synthetic polypeptide free of hybridization withany determined peptidic sequence also possessed by a chloroplast proteinor precursor.

It must be understood that in the preceding definitions "transitpeptide" has the broad meaning indicated hereabove. The transit peptidemay further be selected depending upon the plant species which is to betransformed, although, as mentioned earlier, transit peptides or smallersub-unit precursors containing such transit peptides are often notplant-specific. Sub-unit precursor from one plant species can often beimported and directly processed by the chloroplasts of another.

Preferred DNA sequences encoding a transit peptide for use in therecombinant DNAs of this invention correspond to any of those encoding atransit peptide associated with the small sub-unit of RuBP of pea cells,or also of wheat or soybean cells.

Preferred "first nucleotide sequences" coding for transit peptides aredefined hereafter, merely by way of example. It must be understood thatthe letters above the lines referring to the nucleotide sequence per sedesignate the successive amino acids encoded by the successive tripletsof the nucleotide sequence. The letters below said line correspond todesignations of nucleotides which can be substituted for thosedesignated immediately above them in the nucleotide sequence:

     M   A   S   M   I   S   S   S   A   V   T   T                                  ATG GCT TCT ATG ATA TCC TCT TCC GCT GTG ACA ACA                                -  V   S   R   A   S   R   G   Q   S   A   A   V                             GTC AGC CGT GCC TCT AGG GGG CAA TCC GCC GCA GTG                                             T      T                G                                        -  A   P   F   G   G   L   K   S   M   T   G   F                             GCT CCA TTC GGC GGC CTC AAA TCC ATG ACT GGA TTC                                                     G                                                        -  P   V   K   K   V   N   T   D   I   T   S   I                             CCA GTG AAG AAG GTC AAC ACT GAC ATT ACT TCC ATT                                -  T   S   N   G   G   R   V   K   C                                         ACA AGC AAT GGT GGA AGA GTA AAG TGC                                     

Of course DNA sequences coding for other transit peptides can also beused for the construction of the chimaeric gene of this invention. Forinstance "first sequences" within the meaning of this application mayconsist of a sequence encoding the transit peptide of the lightharvesting chlorophyl a/b-protein complex, normally located in thylakoidmembranes, such as:

     M   A   A   S   S   S   S   S   M   A   L   S                                  ATG GCC GCA TCA TCA TCA TCA TCC ATG GCT CTC TCT                                -  S   P   T   L   A   G   K   Q   L   K   L   N                             TCT CCA ACC TTG GCT GGC AAG CAA CTC AAG CTG AAC                                -  P   S   S   Q   E   L   G   A   A   R   F   T                             CCA TCA AGC CAA GAA TTG GGA GCT GCA AGG TTC ACC                         

The DNA sequence coding for the transit peptide is advantageously thenatural nuclear DNA gene portion or a cDNA obtained from thecorresponding mRNA.

Needless to say that any other DNA sequence encoding similar aminoacidsequences can be substituted therefor. It may for instance becontemplated to use a synthetically produced DNA sequence in which someof the codons differ from corresponding codons in the natural DNAsequence, while nevertheless coding for the same correspondingaminoacids. In that respect the expression "transit peptide" should alsobe understood as extending to any peptide which could differ from anatural transit peptide at the level of some of the aminoacids, to theextent where the substitutions contemplated would not alter theoperability of the resulting peptide to promote the translocation intothe chloroplast of the foreign polypeptide or protein encoded by the DNAsequence associated with or adjacent to the sequence encoding suchpeptide. Thus, the chimaeric genes of the invention may be constructedwith any "first sequence" having substantial sequence homology with anatural DNA sequence encoding a natural transit peptide.

Concerning the protein or polypeptide encoded by the abovesaid "secondsequence", it should also be understood that it may consist of anyprotein or polypeptide sought to be introduced into or processed withinthe chloroplasts of determined plants. Therefore the DNA sequenceencoding it is usually foreign to or heterologous with respect to theDNA sequence encoding the polypeptide or protein normally associatedwith the chosen transit peptide. In other words, the first and secondDNA sequences usually originate from different sources. Particularly thesecond sequence encodes a foreign protein or polypeptide, for instance,of bacterial origin. But, the invention also extends to proteins thatare naturally endogenous to chloroplasts of plants other than the"determined plant" considered hereabove or even to chloroplast proteinscorresponding to natural chloroplast proteins of the same plant, yetdiffering therefrom only by a few amino acids ("mutated" protein).Techniques for directing such mutation (whether in the first or thesecond sequences) are exemplified in a recent paper of S. Gutteridge etal. titled "A site specific mutation within the active site ofribulose-1,5-bisphosphate carboxylase of "Rhodospirillum rubrum" (1984).

Furthermore the chimaeric gene of a preferred recombinant DNA accordingto the invention comprises a promoter region upstream of the abovementioned fused sequences, in such manner that, when said chimaeric geneis inserted within an appropriate vector, the transcription of both theabovesaid first and second sequences are under the control of saidpromoter. The promoter region contemplated hereabove should of course beselected among those which are recognized by the polymerases endogenousto the plant sought to be transformed. Of particular advantage are thepromoters effective in pea, wheat, soybean or tobacco cells. Thepromoter may be that normally associated with the sequence encoding thechosen transit peptide. It may however also be different. An example ofconstruction using another promoter is illustrated later in theexamples. For instance, suitable promoters are those belonging to thegenes of plastocyanin, ferredoxin-NADP+ oxydoreductase, etc. Othersuitable promoters are exemplified in the literature referred to in thisapplication.

Preferably the sequence coding for the transit peptide is under thedirect control of the selected promoter. This means that the firstnucleotide triplet transcribed and expressed under the control of saidpromoter is preferably that of the sequence encoding the transitpeptide. This of course is not critical, for instance, as evidenced bythe first example.

Finally the invention also relates to recombinant vectors, particularlyplasmids which can be introduced and maintained in plant cells andcontaining the abovesaid chimaeric gene, including the above-definedpromoter region.

Preferred vectors of this type are those derived from the Ti-plasmidsreferred to hereabove. More particularly, a preferred vector of thistype comprises in addition to said chimaeric gene a DNA fragmentsuitably positioned with respect to said foreign gene and havingessential sequence homology with the DNA of a Ti plasmid including aT-DNA fragment, the sequences encoding the essential functions capableof causing the transfer of said T-DNA fragment and said chimeric geneinto said plant cells. Particularly, a preferred vector according to theinvention contains a T-DNA border sequence and said chimaeric gene ispositioned close thereto. Even more preferred vectors of this typecomprise two border sequences, the chimaeric gene then being positionedbetween these two border sequences. Concerning general methods forinserting the chimaeric gene in Ti-plasmids, reference is made to thepatents referred to above by way of examples.

Advantageously the recombinant DNA (be it the chimaeric gene as such orthe vector which contains it) should preferably include the appropriatesite for the initiation of the corresponding RNA transcription upstreamof the first condon to be translated, in most cases an ATG codon. It isalso of advantage that the recombinant DNA comprises downstream of theforeign gene to be expressed appropriate transcription termination andpolyadenylation signals.

The invention also concerns a process for achieving and controlling theintroduction of a determined protein or polypeptide (or fragment of saidprotein or polypeptide) into the chloroplasts of determined plant cells.Any suitable process for performing this introduction can be resortedto. Advantageously use is made of vectors of the type exemplified andmodified by a chimaeric gene according to the invention and comprising a"second coding sequence" encoding a protein or polypeptide. But anyother process can be resorted to. For instance a chimaeric geneaccording to the invention can be inserted in plant cells simply by thecalcium chloride polyethylenglyco) precipitation methods or also bymicroinjection into the plant cells.

Additional features of the invention will appear in the course of thefollowing disclosure of the conditions under which the structuralrequirements of vectors capable of transforming plant cells for the sakeof ultimately causing a determined foreign gene product to be insertedin chloroplasts were determined.

In the examples which follow the approach taken has been to construct achimaeric gene encoding a fusion protein containing the transit peptideof the precursor to the small sub-unit of RuBP carboxylase from pea anda coding sequence of the bacterial neomycin phosphotransferase (II)(abbreviated as NPT(II)).

The NPT(II) protein was chosen because NPT(II) protein confersresistance for kanamycin to plants (HERRERA-ESTRELLA et al., 1983:FRALEY et al., 1983; BEVAN et al., 1983), fusion proteins arebiologically active (Reiss et al. 1984b) and an enzymatic assay for insitu detection of NPT(II) or NPT(II) fusion proteins in non-denaturingpolyacrylamide gels has been described recently⁴⁷. This method isparticularly useful to distinguish processed from unprocessed forms ofthe fusion protein.

EXAMPLE I GENERAL OUTLINE

Construction of plasmids pSNIP and pSNIF containing the chimaeric gene(tp-ss-nptII

A genomic clone for one of the rbcS genes from pea was isolated,sequenced and made available by Dr. A. CASHMORE, Rockefeller University,New York (pPSR6). From this clone the promoter signals, (CASHMORE, 1983:HERRERA-ESTRELLA et al., 1984), the first exon coding for the rbcStransit peptide and the first two codons of the mature small subunitprotein, followed by the first intron (83 pb) and part of the secondexon (66 bp) coding for the amino terminus of the mature small subunitprotein were fused via a Sau3A restriction and endonuclease recognitionsite with the BamHI site of the plasmid pKm109/9 (REISS et al., 1984b)which contains the coding region for the nptII gene from Tn5 (BECK etal., 1982).

The fusion gene which was obtained and which contained the transitsequence (56 codons) and 22 codons from the mature rbcS gene linked viaseven artificial codons with the second codon from the nptII gene (FIG.1B) were found to be similarily active. The size of the coding region ofthe nptII gene is 1130 bp. The fusion junction was verified (data notshown) by DNA sequencing (MAXAM and GILBERT, 1977). The chimaericprotein should have a size of Mr 38,023 in the unprocessed and of Mr32,298 in the processed form. Southern type (SOUTHERN, 1975)hybridization data (FIG. 2) established that transformed plant tissuescontained the chimaeric gene constructs in the nuclear DNA and that nodetectable DNA rearrangements had occured during integration. Aschematic representation of the results is given in FIG. 3.

A more detailed disclosure of the construction will be given hereafter,more particularly in relation to FIGS. 1A, 1B and 3.

Production of vectors capable of transforming plants

To introduce the chimaeric genes in the nuclear genomo of plants, theplasmid was inserted into the T-DNA of pGV3851 and of pGV3850, bothderivatives of the Ti plasmid pTIC58, in which parts of the T-DNA wheresubstituted by pBR322 (ZAMBRYSKI et al., 1983; 1984). The T-DNA ofpGV3851 still contains the genes coding for transcripts 4, 6a and 6b(WILLMITZER et al., 1983) which results in a teratoma-like growth of thetransformed tissue (JOOS et al., 1983), whereas all tumor controllinggenes have been eliminated in pGV3850 with the result that plant cellstransformed with this vector can differentiate and grow as normal plants(ZAMBRYSKI et al., 1983; D E BLOCK et al., 1984). The gene constructionswere introduced into pGV3850 and pGV3851 Ti-plasmids by homologousrecombination after mobilization from E. coli to Agrobacterium with thehelp of plasmids R64drd11 and J G28 (VAN HAUTE et al., 1983).

Cointegrates were selected on spectinomycin and streptomycin containingplates and their structure verified by Southern blot hybridization(SOUTHERN, 1975) using various parts of the constructions as probes(data not shown).

Plant transformation

The chimaeric genes were introduced into Nicotiana tabacum cv. Wisconsin38 or SR1 by inoculation of a wounded plantlet or by co-cultivation ofprotoplasts with Agrobacterium. Transformed material obtained bywounding was screened for the presence of nopaline synthase activity(OTTEN, 1982), a cotransferred marker. Transformants (pGV3851::pSNIP)grew on 250 ug/ml kanamycin as green teratoma tissue, suggesting that afunctional chimaeric gene was present and transcribed. In co-cultivationexperiments N. tabacum SR1 protoplasts were incubated with Agrobacteriumcontaining (pGV3850::SNIF) and selected after two weeks with 100 ug/mlkanamycin. From 9 individual colonies which were positive when testedfor NPTII activity, one was chosen and regenerated under constantselected pressure to a fully normal looking plant. Genetic analysisshows inheritance of the NPTII marker in a classical mendelian fashion.These results suggested that transcripts from the chimaeric genes wereproperly processed, transported out of the nucleus and translated into afunctionally active protein.

Light induction of the chimaeric gene

Poly (A)+ and poly (A)-RNA from wild type and from transformed tissues(pGV3851::pSNIP) was isolated and analysed by so called "Northern" gelhybridizations. When the coding region of the nptII gene (BamHI-SmaIfragment from pKM109/9) was used as a probe a complex hybridizationpattern was observed with RNAs ranging between 5,500 nucleotides and8,000 nucleotides in size. These RNAs were detected in light grownteratomas only. Four days of darkness after a day:night rhythm of twelvehours resulted in a marked decrease of the signals (FIG. 4). The verylarge size of these transcripts probably results from the fact that noproper polyadenylation and transcription termination site was introducednear the translation termination signal. No signals of comparable sizeor strength were observed in wild type Wisconsin 38 tobacco or inmaterial obtained from a plant transformed with the pGV3850 vector only(FIG. 4). In order to compare the light dependent transcription of thechimaeric gene with that of both the endogenous rbcS gene and thechloroplast gene coding for the large subunit of Rubisco (rbcL), poly(A)+ and poly (A)-RNA from light- and dark-grown teratoma werehybridized to specific probes for each of these genes. The results areillustrated in FIG. 5A. Signals of the endogenous rbcS transcripts (850nucleotides) were observed at the expected position. Similarly, atranscript of 1750 nucleotides was observed when a rbcL specific probewas used (ZURAWSKI et al.; 1981). The results suggest that the promotorof the rbcL gene, which resides in the chloroplasts, is less sensitiveto light stimuli than both the endogenous rbcS and the newly introducedchimaeric gene. Dot blot experiments were included to quantify theseresults (FIG. 5B). The same probes were used as mentioned before.Individual dots were cut out and the radioactivity counted. A differenceof about 25-fold was measured between poly (A)+ RNA from light- anddark-grown teratoma shoots probed with either rbcS or nptII sequences.In contrast, the difference is only 5-fold for poly (A)- RNA specificfor rbcL sequences. These results support the Northern experimentsindicating that the transcripts of the chloroplast gene coding for thelarge subunit is less sensitive to influence of light in comparison withthe nuclear gene for the small subunit. In addition, it seems that thepea rbcS promotor of the introduced chimaeric gene has a sensitivity todifferent light regimes which is comparable to that of the endogenouspromotor or promotors measured in the tobacco teratoma tissue.

Features of fusion proteins

In order to detect the fusion protein formed between the transitpeptide, the NH₂ -terminal region of the mature small subunit and theNPTII protein in plants, an assay detecting the phosphotransferase-IIactivity in crude extracts of plants was developed. The method wasadapted from published procedures (REISS et al., 1984a) and eliminatesmost of the endogenous self-phosphorylating proteins which interferewith the assay by proteinase K treatment. The results presented in FIG.6 demonstrate that NPTII activity is detected in a crude extract (lane4) of leaves of tobacco plants containing the pGV3850:: pSNIF construct.The activity migrates in the gel assay with a mobility which isintermediate between that of the TP-NPTII fusion protein (35.5 kd) andthat of the normal NPTII PROTEIN (29 kd) from extracts of E. coli (lane1). The relative mobility of the NPTII activity in lane 4 is consistentwith a conclusion that it represents the processed form of the precursorprotein (SS-NPTII) which has a theoretical molecular weight of 32,298.Since the polarity index (CAPALDI and VANDERKOOI; 1972) of the threeproteins is 41 for NPTII, 40 for SS-NPTII and 41 for TP-NPTII, it islegitimate to compare the three proteins by their mobility on nativepolyacrylamide gels (see FIG. 6B). Indeed the unprocessed TP-SS-NPTIIprotein has a molecular weight of about 38,000 and would thereforepresumably migrate more slowly than the TP-NPTII marker, the SS-NPTIIfusion protein is degraded in vitro after isolation yielding activesubfragments with a mobility which approaches that of the normal NPTIIenzyme. That the lower molecular weight spots seen in FIGS. 6A and 7 aredue to unspecific degradation was shown by demonstrating that this andother NPTII fusion proteins are actually degraded in vitro in bothbacterial and plant extracts (data not shown). Incubation in thepresence of protease inhibitors could not completely prevent thisdegradation. No activity was detected in control extracts from tobaccolacking the TP-SS-NPTII chimaeric gene (lane 3). The SS-NPTII activityobserved in crude extracts can also be detected in isolated chloroplasts(lane 2). The relative amount of activity detected in the chloroplats issignificantly less than the activity observed in crude extracts. This isprobably due to leakage of the activity out of the chloroplasts duringchloroplast isolation. Indeed the procedure used to isolate chloroplastsled, with this particular plant material, to a substantial damage of thechloroplasts. More than 90% of the chloroplast material is eithervisibly damaged or runs at a reduced density in the percoll gradients.Further manipulations during recovery and concentration prior to theNPTII assay could contribute to further minor damage leading tosignificant loss of the protein by leakage. These observations do notexclude the possibility that although all of the precursor TP-SS-NPTIIprotein is processed to the SS-NPTII form, it is not actually alltransported in vivo into the stroma of the chloroplasts. However thedata obtained clearly demonstrate that at least some of the processedSS-NPTII protein is within the stromal fraction of the chloroplasts.Indeed the activity associated with the chloroplasts was shown to belocated within the stroma by demonstrating that broken chloroplasts didnot contain any detectable NPTII activity and that the NPTII activity inintact chloroplasts could not be eliminated by trypsin treatment (datanot shown). Further evidence that the detected SS-NPTII activity wasderived from the introduced light inducible chimaeric gene was obtainedby demonstrating that the activity was significantly reduced whentobacco plants containing the pGV3850::pSNIF construct and grown in thegreen house in a 12 hour light/dark regime (FIG. 7, lane 3) weretransferred for 96 hours to complete darkness (FIG. 7, lane 2).

The details concerning the conditions under which the constructions ofDNA recombinants were obtained and the methods used for appreciating theresults asserted hereabove, inasmuch as they are not ascertainable fromthe previous discussion will be recalled hereafter.

MATERIALS AND METHODS

Strains and plasmids

E. coli DH1 was used for in vitro transformation. Agrobacterium C58CIRifwas the receptor strain in all bacterial conjugations. The conjugationfollowed the protocol described by Van Haute et al. (1983) and ZAMBRYSKIet al. (1984).

DNA techniques

Restriction endonucleases and other DNA modifying enzymes were used asrecommended by the manufacturers. Other techniques were used asdescribed by MANIATIS et al. (1982).

Nopaline assay

The presence or synthesis of nopaline due to expression of the nos genein transformed calli and regenerating shoots from these calli wasmonitored according to OTTEN (1982).

Plant transformation

Small axenically growing plants were kept in 1/2 M+S medium (MURASHIGEand SKOOG, 1962) in jars and were inoculated after decapitation withAgrobacterium strains as described (ZAMBRYSKI et al., 1984). Wound calliwere removed and put on medium containing 0.2 mg/l benzaminopurine and0.6 mg/l indolacetic acid and 0.5 mg/ml cefotaxime (HOECHST). After ca.4 weeks the callus material was transferred to hormone free medium andemerging shoots were tested for nopaline production. Nopaline synthasepositive shoots were propagated and tested on 100 to 500 ug/mlkanamycin. Teratoma shoots which grew on concentrations of 100 ug/ml orhigher were used for analysis. Protoplast were kept in coculture withAgrobacteria according to MARTON et al. (1979) with modificationsdescribed by HAIN et al. (1985).

Analysis of DNA and RNA

DNA was isolated according to BEDBROOK (1981) from preparations ofnuclei. The DNA was digested with restriction endonucleases (10-30ug/lane, overnight digestion with a 3-fold excess of enzymes), separatedon agarose gels according to size and transferred to nitrocellulosefilters (THOMAS, 1983). Hybridization with radioactive probes wasperformed in 50% formamide, 4 times SSC, 10 times Denhardt's solution,0.2 SDS and 0.1 mg/ml calf thymus DNA at 50° C. for 48 hours (BOHNERT etal., 1982). The filters were washed twice for 15 minutes each in 50%formamide, 4 times SSC at the hybridization temperature, followed bywashing in 50% formamide, 3 times SSC at room temperature (1-4 hours)and 2 times SSC at room temperature (1 hour). Dot blot hybridizationswere performed according to THOMAS (1983) with DNA amounts covering arange equivalent from 1000 to 0.1 gene copies per sample. Hybridizationwas as described above. RNA was isolated according to CHIRGWIN et al.(1979), and separated into poly (A)+ and poly (A)-RNA by passage overoligo d(T)-cellulose (Collaborative Research, type III) following theprocedure of AVIV and LEDER (1972). RNAs were separated according tosize in 1% agarose gels containing 5 mM methylmercury hydroxide (BAILEYand DAVIDSON, 1976). Hybridizations with ³² P-labelled, nick-translatedprobes were carried out as described (BOHNERT et al., 1982); between 2and 3×10⁶ cpm/lane were used.

Neomycin phosphotransferase activity assay

The assay was adapted for plant extracts from a procedure worked out forbacterial and animal cell lysates (REISS et al., 1984a). Between 20 and100 mg of tissue from transformed plants was crushed in 0.1 ml buffer(10% glycerol, 5% ∞-mercaptoethanol, 62.5 mM tris/HCl, pH 6.8, 50 ug/mlbromophenol blue and 0.1% SDS). Several protease inhibitors were used inan attempt to inhibit specific and unspecific proteases. Aprotinin(Trade name Trasylol) was used at a final concentration of 100 ug/ml inwater. p-hydroxy-mercuri-benzoate (PHMB) was used at a concentration of1 mM, ε-amino-n-caproic-acid and 1-10-phenantroline were added to afinal concentration of 5 mM. Protease inhibitors were used according toGray (1982). Cristalline phenylmethylsulfonylfluoride (PMSF) was addedimmediately before use at a concentration of 100 ug/ml. The clearedhomogenate (5 min., 13,000 rpm, Eppendorf centrifuge) was loaded onto10% non-denaturing polyacrylamide gels (Laemmli, 1970; without SDS).After electrophoresis the buffer in the gel was exchanged against 67 mMTris/maleate, 42 mM MgCl2, 400 mM NH4Cl, pH 7.1, and the acrylamide gelwas covered by an agarose gel (1%) containing kanamycin-sulfate (1mg/ml) and γ³² P-ATP (5 uCi/um pf a specific activity of 2000-3000Ci/mMol in the same buffer as the polyacrylamide gel. The gel-sandwichwas covered by Whatman P81 paper, Whatman 3MM paper, and paper towels.After 3 hours the P81 paper was incubated for 30 minutes in a solutioncontaining 1% SDS and 1 mg/ml proteinase K in water at 60° C. andsubsequently washed several times in 10 mM phosphate buffer (pH 7.5) at80° C., dried and exposed to Kodak XR5 film for up to 48 hours. Theprinciple of this method is the binding of kanamycin to thephosphorylated DEAE paper by which the positions in the gel are revealedwhere a kanamycin phosphorylating activity migrated. The additionalproteinase treatment suppresses signals of plant activities which afterphosphorylation bind to P81 paper but do not phosphorylate kanamycin.

Isolation of chloroplasts

Chloroplasts were isolated from 1-2 g of leaves of transformed plants.Structurally intact chloroplats were collected from Percoll (Pharmacia)gradients (Ortiz et al., 1980). The washed chloroplasts wereconcentrated by centrifugation, lysed and than used for the in situdemonstration of NPTII activity as described above. Trypsinisation ofchloroplasts was performed according to BARTLETT et al. (1982).

CONSTRUCTIONAL DETAILS AND METHOD EMBODIMENTS IN RELATION TO THEDRAWINGS

1) Construction of the chimaeric rbcS-npt-II genes pSNIP and pSNIF (FIG.1A).

A BamHI-SalI fragment from pKM109/9 REISS et al., 1984b) containing theentire coding region from a modified nptII gene from Tn5 (BECK et al.,1982) was inserted in plasmid pPSR6 Δ-RV next to a 950 bp DNA fragment(EcoRI-EcoRV) containing the promotor region and the 5'-end of the rbcSgene resulting in plasmid I-22. In this plasmid the HindIII-BamHIfragment was replaced by a HindIII-Sau3A fragment (53 bp) from theoriginal rbcS clone (pPSR6) to form the plasmid II-4 containing thefusion gene. The pBR derived region in II-4 was exchanged against anEcoRI-SalI fragment from pGV710 in order to introduce streptomycin andspectinomycin resistance to be used as a marker to select forcointegration of this final plasmid (pSNIP (10.4 kbp)) with theTi-plasmid in Agrobacterium, Plasmid pSNIF (12.3 kbp) was constructed byreplacement of the SmaI-SalI fragment of pSNIP with an PvuII-XhoIfragment from the octopine synthase gene from plasmid pAGV40(HERRERA-ESTRELLA et al. 1983; DE GREVE et al., 1983) harboring thepolyadenylation site of that gene next to a BamHI restriction site ofthat gene next to a BamHI restriction site.

2) Structure of the rbcS-npt-II chimaeric gene (FIG. 1B).

The black bar represents the transit-peptide sequence with the firstATG, the white area (two codons in exon 1 and 22 codons in exon 2) isinterrupted by the first intron and represents the mature rbcS sequence.The hatched part represents the nptII sequence.

3) Southern hybridization experiments (FIG. 2).

Hybridization of different probes to nuclear DNA from transformed(pGV3851::pSNIP) (a, c and e) and untransformed (b and d) tobacco. InSouthern hybridization experiments (Southern, 1975) lane a and b resolvebands of different size resembling the small subunit gene family when a661 bp EcoRV-AvaIII DNA fragment from the genomic small subunit clonewas used as probe (Cashmore, 1983). An additional band of 10.4 kbpreveals the chimaeric gene fragment in lane a. In lanes c, d and e DNAwas digested with PstI and EcoRI and either the promoter region of thesmall subunit gene (972 bp EcoRI/HindIII fragment) (lane c and d) or thecoding region of the nptII gene (1000 bp BamHI/SmaI fragment fromplasmid pKM109/9) were used as probes. In lane c a strong signal isdetected from untransformed material (lane d). Weak signals in lane care most likely due to crosshybridization of endogenous rbcS sequencesor incomplete digestion of the DNA. In lane e a band of 0.9 kbp lightsup the internal PstI fragment of the nptII gene and the weaker bandshows again the 1.5 kbp fragment seen in lane c, due to a small overlapbetween the probe and the promotor region of the chimaera.

4) Schematic representation of the organization of the fusion and theflanking vector sequences (FIG. 3).

Sizes are indicated by kbp. the chimaeric rbcS-nptII coding region isindicated by an open bar, the 5'-flanking sequence by a closed bar.EcoRI and PstI indicate restriction endonuclease sites. SpR and ApRrepresent antibiotic resistance markers against spectinomycin andampicillin. Numbers indicate the size of fragments obtained in theSouthern experiments (FIG. 2). The DNA fragments between the gene fusionand the right part of the T-DNA represent the pBR322 sequences presentin the vector pGV3851.

5) Transcriptional activity of rbcS promotor (FIG. 4).

RNA was separated in denaturing 1% agarose gels and transferred tonitrocellulose filters which were probed with different parts of theconstruction. The coding region of the nptII gene (BamHI-SmaI fragmentfrom pKM109/9) was used as a probe. Lane 1: RNAs from light grownteratoma shoots. Lane 2: RNAs from plant material kept in darkness forfour days after a day/night rhythm of twelve hours. Lane 3: RNAs fromplant leaves transformed with pGV3850. Lane 4: RNAs from wild typeWisconsin 38. Weak signals in the latter are probably due tocontaminating material in the probe which hybridizes to mRNA promotoractive in the T-DNA or near the position of insertion in the plantchromosome. Numbers on the left indicate size in nucleotides, numbers onthe right refer to the Svedberg values of RNA markers.

6) Comparison of light dependence of rbcS and rbcL promotors (FIG. 5A).

Poly (A)+ RNA from teratoma shoots grown in a daily rhythm of 12 hourslight/dark (L) and material kept subsequently for four days in the dark(D) were hybridized to an nptII specific probe (see FIG. 4) and to arbcS specific probe (see FIG. 2). The endogenous rbcS transcripts areobserved at the position of 850 nucleotides. Poly(A)-RNA was analysedwith the same technique probed with a 1750 bp fragment from a rbcL gene(ZURAWSKI et al., 1981). Numbers on the left refer to Svedberg values ofRNA markers or to the size of the mRNA (right).

7) Dot Blot Hybridization to RNA From Transformed (pGV3851::pSNIP) PlantMaterial (FIG. 5B)

L indicates light grown material in 12 hour light/dark cycle. Dindicates subsequent growth in the dark for four days. Single dots wherecut out and radio-activity measured.

8) Demonstration of Transport of TP-SS-NPTII Precursor in Chloroplastsof Tobacco Plants Containing the pGV3850::pSNIF Construct (FIG. 6A)

The results obtained in each lane are commented hereafter:

Lane 1: extracts from E. coli pGLT neo1 expressing a TP-NPTII protein(VAN DEN BROECK et al., Nature in press) in E. coli pKM2 containing theTN5 encoded NPTII enzyme.

Lane 2: Neomycinphosphotransferase activity in chloroplasts purifiedfrom leaves of tobacco plants containing the chimaeric tp-ss-nptII gene.

Lane 3: Crude extract from leaves of a control SR1 tobacco plant.

Lane 4: Crude extract from leaves of tobacco plants containing thechimaeric tp-ss-nptII gene. The P.K. band is presumed to be due to acytoplasmic self-phosphorylating protein and C.P.K. is presumed to bedue to a chloroplast self-phosphorylating protein.

9) Graphic Display of the Relative Mobility of the Different NPTIIActivities Detected in FIG. 6A (FIG. 6B)

As described hereabove it is legitimate to make the assumption thatthese proteins are separated according to molecular weight on thesenative gels because of their very similar polarity index (CAPALDI andVANDERKOOI; 1972).

10) Light Dependant Expression of the SS-NPTII Fusion Protein (FIG. 7)

Lane 1: Idem as for FIG. 6A.

Lane 2: Idem as for FIG. 6A lane 4 except for the fact that the plantswere kept in complete darkness for 96 hours before extraction. Lane 3:Idem as for FIG. 6A. lane 4.

The results obtained demonstrate that the use of Agrobacterium vectorsto transfer and express genes in plant cells (amply documented by CAPLANet al., 1983; ZAMBRYSKI et al., 1983; 1984; HERRERA-ESTRELLA et al.,1983; 1984) can be extended to target a foreign protein for a specificcell compartment, namely the chloroplast. The results furtherdemonstrate

(i) that the gene fusion is integrated in the nuclear DNA of tobaccowithout rearrangement of the DNA

that the transcription of this chimaeric gene (which contained a lightinducible promoter sequence) is regulated by light.

It is important to note that the induced transcription of thisintroduced gene is as efficient as that of the endogenous small subunitgene(s) and rather more efficient than previously observed in tobaccowith another chimaeric gene using the same pea small subunit promoter(HERRERA-ESTRELLA et al., 1984). Possibly the higher level of inducedsteady state mRNA in these tissues is due to improved mRNA stability.The presence of one intron in the transcript derived from this transitpeptide small subunit neomycin phosphotransferase chimaeric gene(tp-ss-nptII) and the absence of any intron in the constructiondescribed by HERRERA-ESTRELLA et al., (1984), might explain an increasedstability of this RNA (Hamer and Leder, 1978). Our observations alsodemonstrate that the pea small subunit promoter can be active in leavesof normal tobacco plants. This is in contrast to previous observationsin several laboratories which indicated that the pea small subunitpromoter while active in tobacco tissue cultures and teratomas, wasinactive in leaves of normal plants. Possibly a position effect isinvolved in this phenomenon, the chimaeric tp-ss-nptII gene in(pGV3851::pSNIP) did not contain a polyadenylation or a transcriptiontermination signal, which probably explains the observed very largetranscripts. It will be shown in Example II that the provision of asuitable polyadenylation or a transcription termination signal at theappropriate location after the nptII gene results in the production oftranscripts having substantially the same lengths as the transcripts ofthe nptII in its natural environment.

The data obtained hereabove demonstrate that the chimaeric tp-ss-nptIIgene, which upon expression yields a fusion protein with a transitpeptide and the conserved amino acid sequence flanking the processingsite, is indeed translocated to the chloroplasts and is processed toyield a fusion protein located in the stroma, consisting of the NH₂-terminal end of the small subunit protein and an active NPTII protein.This SS-NPTII fusion protein migrates in the gel NPTII-assay with anelectrophoretic mobility which is intermediate between the TP-NPTII(35.5 kd) and that of the original NPTII activity (29 kd). This mobilityis in very good agreement with the molecular weight (32,298) of theSS-NPTII fusion protein. The results obtained indicate that this fusionprotein, which confers kanamycin resistance to the transformed tobaccoplants, is located within the chloroplasts and might leak out when thechloroplasts are broken.

However, the results obtained with the construction described in ExampleII hereafter demonstrate that the NPTII component of a precursor proteinwhich contains only the transit peptide sequence immediately fused tothe NPTII protein and thus missing part of the conserved aminoacidsequence flanking the processing site, is equally translocated acrossthe chloroplast envelope and apparently properly processed. The latterresults indicate that the transit peptide sequence alone is sufficientto both transport and process precursor proteins into chloroplasts.

EXAMPLE II

In this example a chimaeric gene encoding a fusion protein consisting ofthe transit peptide of the precursor to the small subunit of RuBPcarboxylase from pea⁴⁴ directly linked to the amino-terminus of NPT(II)was constructed.

In other words the bacterial enzyme into a novel "precursor" polypeptidewas tested for its ability to be post-translationally imported andprocessed by chloroplasts both under in vivo and in vitro conditions.

General Outline of the Plasmids Construction

Two plasmids have been constructed which contain chimaeric genesencoding TP-NPT(II) (FIG. 8A). In the first plasmid, pGSSTneo3, thecoding sequence for TP-NPT(II) is under control of the pea ss3.6promoter which directs expression of chimaeric genes in plantcells⁴²,45. This construction has been used to study the fate of thefusion protein in vivo in transformed tobacco cells. Another plasmid,pGLTneo1, was constructed to direct the synthesis of TP-NPT(II) in E.coli under control of the lacUV5 promoter⁴⁵ in order to obtainsufficient quantities of the fusion protein for use in in vitroreconstitution experiments with isolated chloroplasts. The fusionprotein encoded in both plasmids consists of the 57 amino acid transitpeptide and the first methionine of the mature small sub-unitpolypeptide encoded by the pea ss3.6 gene⁴⁴, a 7-aminoacid linkerfragment, and the NPT(II) devoid of the first methionine⁴⁵ (263aminoacids). The amino acid sequences in the authentic small sub-unitprecursor encoded by ss3.6 and the fusion protein are compared in FIG. 9lane 2. It can be seen that the Cys/Mot cleavage site of the precursorto the small sub-unit is left intact in the TP-NPT(II) fusion protein.

To study the fate of the TP-NPT(II) fusion protein in vivo, it wasnecessary to first obtain transformed plant cells expressing thetp-npt(II) gene product.

The tp-npt(II) gene of pGSSTneo3 was brought into the genome of plantcells by means of the vector pGV3851, a derivative of the AgrobacteriumTi-plasmid pTiC58⁴⁸. The plasmid pGV3851 contains a deletion whichremoves several of the T-DNA-encoded transcripts, including thoseinvolved in auxin production, but retains the gene involved in cytokininsynthesis. The result of this modification is that Agrobacteriumharbouring pGV3851 induces shoot-forming tumours. In pGV3851, thedeleted portion of the T-DNA has been replaced by pBR322. pGSSTneo3 wasinserted into the T-DNA of pGV3851 by recombination through the pBR322homology⁴⁹.

The T-DNA of several Agrobacterium exconjugants obtained onkanamycin-containing plates was examined by Southern hybridizationanalysis⁵⁰ to confirm that the proper cointegration between pGSSTneo3and the T-DNA of pGV3851 had occurred. The results obtained for one ofthese pGV3851::pGSSTneo3 exconjugants is shown in FIG. 9, lane 1.

Stems of sterile tobacco seedlings were inoculated with this strainafter wounding with a needle below the first internode. After 2-3 weeks,green, shoot-forming tumours appeared. Axenic tissue was obtained bygrowing the transformed tissue in vitro on Murashige and Skoog (MS)medium⁵² containing 500 μg/ml of cefotaximum, an antibiotic to whichampicillin-resistant agrobacteria are sensitive. During propagation ofthe tissue, the sucrose concentration of the MS medium was reduced from3% to 1% to improve greening. The green tissues were able to grow onmedium containing 200 μg/ml of kanamycin, indicating that the tp-npt(II)gene was present and functionally expressed. The presence of thetp-npt(II) gene was confirmed by Southern hybridization analysis⁵⁰ ofgenomic DNA obtained from the transformed callus tissue (FIG. 8B).

A parallel series of cointegration and transformation experiments (datanot shown) provided tobacco tumours containing a second chimaeric gene,nos-npt(II)^(ref) 40 coding for the unaltered NPT(II) protein undercontrol of the promoter from the nopaline synthase gene³⁵,43. Thisallowed the study the fate of NPT(II) itself in transformed cells.

Fate of the tp-npt(II) Gene Product in Plant Cells

Since the TP-NPT(II) fusion protein is not a normal component of plantcells, it was of interest to determine the final location of the fusionprotein in transformed cells. Specifically, we wished to know whetherthe transit peptide alone is capable of directing the uptake andprocessing of the TP-NPT(II) fusion protein by chloroplasts in vivo.Therefore, the following series of experiments were performed todetermine the fate of both TP-NPT(II) fusion protein and unalteredNPT(II) in transformed tobacco cells.

The presence of NPT(II) or active NPT(II) fusion proteins in a givenextract can be determined using an in situ enzymatic assay forphosphotransferase activity after gel electrophoresis (FIG. 10). Thepositions of the original NPT(II) and the TP-NPT(II) fusion protein weredetermined by assaying extracts of E. coli harbouring either pBR322::Tn5or pGLTneo1, prepared as described⁴⁷. As shown (lane 3, FIG. 10), theenzymatic assay on extracts of plant tissue that does not contain theNPT(II) coding sequence in its genome reveals two bands ofphosphotransferase or kinase activity (these are noted by P.K., plantkinase). These bands do not represent NPT(II) activity since they canalso be observed when no kanamycin is included as substrate in theenzymatic reaction (data not shown). The faster migrating band is alsofound with chloroplast preparations from the same tissue (lane 4, FIG.10). When a bacterial extract containing the TP-NPT(II) fusion proteinencoded by pGLTneo1 is mixed with plant extract, a new major band of NPTactivity appears (lane 2, FIG. 10). This band migrates more slowly thanNPT(II) encoded by Tn5 (lane 1, FIG. 10), and probaby corresponds to thebona fide TP-NPT(II). The change in mobility is due to a change in bothmolecular weight and charge as a result of the addition of the transitpeptide. In lane 2 (FIG. 10), also minor bands with higher mobility canbe observed. These likely correspond either to degradation products ofthe fusion polypeptide, or to smaller polypeptides translated from aninternal ATG of the TP-NPT(II) coding sequence.

Crude extracts obtained from transformed tissue containing a nos-npt(II)chimaeric gene contain NPT(II) activity (lane 5, FIG. 10). However,intact chloroplasts isolated from the same tissue do not have detectableNPT(II) activity associated with them (lane 5, FIG. 10). Thisobservation suggests that the product of this chimaeric gene lacks theinformation necessary to mediate its translocation into chloroplasts.Crude extracts from tissue containing the tp-npt(II) chimaeric gene alsocontain considerable NPT(II) activity (lane 7), FIG. 10). When intactchloroplasts are isolated from this tissue, considerable levels ofNPT(II) activity are found to be associated with them (lane 8, FIG. 11).Moreover, the one neomycin phosphorylating protein observed in both thecrude extract and the isolated chloroplats, migrates with the samemobility as the Tn5 authentic protein, and differs from the NPT(II)fusion protein from E. coli harbouring the tp-npt(II) chimaeric gene(see also FIG. 11, lanes 1, 2, 3). Even after longer exposure of theauto-radiogram there was no indication of the presence of this NPT(II)fusion protein. These observations show that the NPT(II) activity isconcentrated in the chloroplast fraction, and that the TP-NPT(II) fusionprotein is cleaved very efficiently close to the fusion site, removingthe transit peptide.

Since the mature SS polypeptide is part of a soluble protein present inthe stroma, it was of interest to determine whether the NPT(II) activityassociated with the isolated chloroplast fraction is also sequestered inthe same suborganellar compartment. Therefore, chloroplasts frompGV3851::pGSSTneo3-transformed tissue were lysed by resuspension in ahypo-osmotic buffer, and fractionated into stromal and membranefractions. The membrane fraction was further washed to eliminate stromalcontamination. Aliquots from these fractions were then subjected toelectrophoresis on non-denaturing gels and assayed in situ for NPT(II)activity. The results of this analysis (FIG. 11) clearly demonstratethat all of the enzyme activity associated with the chloroplast fractionisolated from transformed tissue is located in the stromal (lane 3, FIG.11) rather than membrane (lane 4, FIG. 11) fraction of the plastids. Toensure that these findings represent uptake of the fusion protein by thechloroplasts and not non-specific binding to the plastid envelope andrelease during organelle fractionation, aliquots of isolatedchloroplasts were subject to protease treatment⁵³. Equal amounts ofchloroplasts from protease-treated and non-treated preparations werethen fractionated as described above, and stromal fractions assayed forNPT(II) activity. A large percentage of the NPT(II) activity present innon-treated chloroplasts (lane 3, FIG. 12) remains present inprotease-treated chloroplasts (lane 4, FIG. 12) until these chloroplastsare broken (lane 2, FIG. 12). the slight decrease in activity observedis likely the result of losses from plastid lysis rather than the lackof sequestering of the processed fusion protein within the chloroplast.

These results, clearly demonstrate that the TP-NPT(II) fusion protein istargeted to the chloroplast, translocated into the stroma, and processedin a fashion similar to that of the small subunit polypeptide.

In vitro Uptake and Processing of the Fusion Protein by IsolatedChloroplasts

As an alternative approach to determine whether the transit peptidealone is sufficient to direct post-translational uptake of proteinsother than the mature small subunit polypeptide into chloroplasts and totest whether chloroplasts can recognize and proteolytically remove thetransit peptide of a fusion protein, a series of in vitro reconstitutionexperiments were carried out with isolated intact chloroplasts. The invitro approach has previously been shown to be useful in the analysis ofchloroplast translocation processes⁶,10-17. Here, we have adapted thismethod for use with fusion proteins produced by E. coli.

Bacterial extracts containing the TP-NPT(II) fusion protein wereprepared by sonication of exponentially growing liquid cultures ofEscherichia coli harbouring pGLTneo1. Aliquots of the TP-NPT(II)containing cleared bacterial extracts were incubated for 1 hour withchloroplasts isolated from pea leaves⁵³. Following incubation, thechloroplasts were reisolated form the incubation mix and washed severaltimes an isosmotic buffer until no TP-NPT(II) activity was detected inthe supernatant.

This preparation was used to determine whether there was NPT(II)activity associated with the stroma or membrane fraction of thesechloroplasts. Lanes 1 and 2 or FIG. 6 show respectively the position ofNPT(II) and TP-NPT(II) present in bacterial extracts. Lane 3 (FIG. 6)shows that prior to incubation with E. coli extracts containingTP-NPT(II), the stroma of chloroplasts isolated from pea does notcontain any phosphotransferase or kinase activity comigrating witheither the TP-NPT(II) fusion polypeptide or authentic NPT(II). However,as observed earlier in tobacco, our assay conditions reveal anendogenous kinase activity (P.K.) associated with chloroplasts. Afterincubating these chloroplasts with bacterial extracts containingTP-NPT(II), the stromal fraction obtained from the isolated organellescontains a considerable level of NPT(II) activity (lane 4, FIG. 6),whereas the membrane fractions does not (lane 6, FIG. 6). This NPT(II)activity migrates like the original bacterial enzyme, which indicatesprocessing. To confirm that the NPT(II) activity observed in the stromalfraction of chloroplasts incubated in the presence of the TP-NPT(II)fusion protein was the result of uptake and not the result of liberationduring the fractionation procedure of protein bound to the chloroplastenvelope, chloroplasts were reisolated from the uptake incubationmixture, washed and subjected to limited proteolysis⁵³. Followingrepurification, protease-treated chloroplasts were fractionated asabove, and the NPT(II) activity was determined in both the stromal andmembrane fractions. Most of the stromal NPT(II) activity appears to beprotected against protease digestion since the amount of activityrecovered (lane 55, FIG. 6) is similar to that found in non-treatedchloroplasts (lane 4, FIG. 6). The membrane fraction of protease-treatedchloroplasts was completely free of activity (lane 7, FIG. 6). Similarresults on in vitro uptake of the TP-NPT(II) fusion protein have beenobtained using intact chloroplasts isolated from young expanding tobaccoleaves (data not shown).

These results demonstrate that the transit peptide of the precursor tothe small subunit of ribulose-1,5-bisphosphate is capable of mediatingthe uptake of polypeptides other than the mature small subunit bychloroplasts under in vitro assay conditions. That uptake of the NPT(II)protein by chloroplasts in vitro does not occur in the absence of thetransit peptide (data not shown) is consistent with our in vivoobservation that chloroplasts prepared from callus tissue transformedwith nos-npt(II) do not contain activity. These observations furtherconfirm the requirement for the transit peptide in the translocationprocess.

Unlike previous in vitro uptake studies⁶, 11-17 which relied on the useof wheat germ extracts for the synthesis of precursor, polypeptides, wehave used an E. coli expression system for the preparation of our fusionprotein. Since translocation of the fusion protein proceeds in this invitro uptake system, this may be taken as an evidence for the lack of arequirement for additional cytoplasmic factors in the translocationmechanism. However, in contrast to translocation studies with microsomalmembranes⁵⁴, it is not practical to wash chloroplast preparations withhigh salt buffers. Consequently, we cannot completely eliminate thepossibility that the translocation of chloroplast proteins requiresadditional cytoplasmic factors which may be tightly bound to ourchloroplast preparations.

1-Detailed Description of the Construction of Plasmids ContainingChimaeric Genes Encoding the TP-NPT(II) Fusion Protein (FIG. 8A)

A 1-kb EcoRI-SphI restriction fragment from pPSR6, a pBR327 derivativecontaining the pea small subunit ss3.6 gene⁴⁴, was purified from a 1%agarose gel. This fragment, which contains the promoter region,nucleotide sequences encoding the transit peptide and first methioninecodon of the mature small subunit polypeptide, was ligated intoEcoRI/BamHI-cut pKm109/9 to replace the small EcoRI/BamHI fragment infront of the NPT(II)-coding region. The plasmid pKm109/9 is a pBR322derivative containing the npt(II) gene of Tn5 devoid of its5'-untranslated region and the first methionine codon⁴⁵. To fuse the3'-overhanging end of the SphI restriction site with the 5'-overhangingend of the BamHI restriction site, a single-stranded oligonucleotide 5'GATCCATG 3', complementary to both protruding ends, was synthesized andadded to the ligation mix⁵⁵. After fusion, the SphI site is abolished,but the BamHI site remains. The resulting plasmid, pGSSTneo1, wasrestricted with SmaI and a 700-bp PvuII fragment, containing thetranscription termination and polyadenylation signal from the ocsgene⁵⁶, was ligated into the site to ensure proper 3' transcriptiontermination and processing. The intermediate pGSSTneo2 plasmid was thenused in two different cloning steps.

(A) A 1,400 bp BamHI fragment from pUC4K^(ref).57 encoding the kanamycinresistance gene from Tn903 was isolated and cloned into the unique BglIIrestriction site of pGSSTneo2 yielding the plasmid pGSSTneo3. Kanamycinresistance is used as a marker to select for the cointegration ofpGSSTneo3 with the Ti-plasmid in Agrobacterium.

(B) A 200 bp EcoRI/HindIII fragment from pKm109/3^(ref).45 containingthe lacUV5 promoter region, was exchanged for the small EcoRI/HindIIIfragment of pGSSTneo2. This allows for the expression of the TP-NPT(II)fusion protein in E. coli. The resulting plasmid is referred to aspGLTneo1. Abbreviations: Ap^(R), ampicillin resistance; Km^(R),kanamycin resistance. Symbols: ------, pBR322 sequence; [[[neo, codingregion for NPT(II); [, lacUV5 promoter region; Δ, 3' end. Representationof the octopine synthase gene: [Pocs, promoter region; [ocs codingregion. Representation of the gene for the small subunit ofribulose-1,5-bisphosphate carboxylase: [ , promoter and 5'-untranslatedregion; [///, sequence encoding the transit peptide; [, exon; [, intron.

2-Partial Aminoacid Sequence Comparison of the TP-NPT(II) Fusion Proteinand the Precursor to the Ribulose-1,5-bisphosphate Carboxylase SmallSubunit Polypeptide (FIG. 8B)

Partial aminoacid sequences for the precursor to the small subunitpolypeptide or ribulose-1,5-bisphosphate carboxylase encoded by the peass3.6 gene⁴⁴ (upper line) and the TP-NPT(II) fusion protein (lower line)are presented. The area near the processing site of the small subunitprecursor and the fusion point for the TP-NPT(II) fusion protein areshown. The arrow indicates the protoolytic processing site defined forthe small subunit precursor. The amino acid residues derived from theoriginal NPT(II) protein are underlined. Amino acid residues arenumbered above the sequences with the first methionine residue of themature small subunit protein being taken as aminoacid number 1.

Met . . . Ser Asn Gly Gly Arg Val Lys Cys Met Gln Val Trp Pro Pro IleGly Lys Lys . . .

Met . . . Ser Asn Gly Gly Arg Val Lys Cys Met Asp Pro Ala Asn Leu AlaTrp Iso Glu

3) Incorporation of tp-npt(II) Gene Into the Genome of Plant Cells

To insert pGSSTneoIII in between the PGV3851 T-DNA borders, pGSSneoIIIwas first introduced into the E. coli strain Gj23 which harbours thehelper plasmids R64drdII and Gj20. These last two plasmids provided theTra and Mob functions required to mobilize pGSSTneo3 from E. coli toAgrobacterium tumefaciens (harbouring pGV3851). Thus, after conjugationbetween the corresponding E. coli and A. tumefaciens strains,Agrobacterium exconjugants the cointegrate between pGSSTneoIII andpGV3851 were selected on kanamycin containing plates.

The T-DNA of several kanamycin-resistant Agrobacterium exconjugants wasexamined by Southern hybridization analysis⁵⁰ to confirm that the propercointegration between pGSSTneo3 and the T-DNA of pGV3851 had occured.The result obtained for one of these pGV3851::pGSSTneo3 exconjugants isshown in FIG. 3.

Reference is also made at the more detailed description of FIG. 10 whichappears hereafter.

4) Southern Hybridization Analysis of Agrobacterium and Plant DNA (FIG.9)

The autoradiogram above shows the results of Southern hybridization⁵⁰analysis confirming the presence and the structure of the tp-npt(II)chimaeric gene in both cointegrate pGV3851::pGSSTneo3 DNA and in genomicDNA from transformed tobacco cells. Lane 1, total Agrobacterium DNA frompGV3851::pGSSTneo3; lane 2, plant genomic DNA from tobacco callustransformed with pGV3851::pGSSTneo3. In both lanes two fragmentshybridize with the specific probe: one fragment of 2.6 kb representingthe EcoRI/BamHI fragment of pGSSTneo3 containing the Km^(R) gene ofTn903 and the SS promoter and transit peptide region; a second fragmentof 1.85 kb representing the BamHI/SalI fragment of pGSSTneo3 thatcontains the coding region of NPT(II) and the OCS 3' end.

Total Agrobacterium DNA⁵⁸ and plant genomic DNA from transformed callustissue⁵⁹ were prepared and restricted with EcoRI, BamHI, and SalI.Digest products were fractionated on a 1% agarose gel, transferred tonitrocellulose paper, and hybridized with a ³² P-labelled probe specificfor the promoter and the coding region of the TP-NPT(II) fusion protein(the probe was the smaller EcoRI/SalI fragment of pGSSTneo1; see FIG.8A).

5) Localization of NPT(II) Activity in Chloroplasts Callus Tissue (FIG.10)

The autoradiogram shows the presence and mobility of NPT(II) activity inbacterial extracts and cellular fractions following in situ localisationon a 10% non-denaturing polyacrylamide gel⁴⁷. Lane 1, E coli extractscontaining NPT(II), mixed with crude extract of greenpGV3851-transformed tobacco tissue; lane 2, E. coli extract containingTP-NPT(II), mixed with crude extract of green, pGV3851-transformedtobacco tissue; lane 3, crude extract from green pGV3851-transformedtobacco tissue; lane 4, intact chloroplasts from greenpGV3851-transformed tobacco tissue; lane 5, crude extract from greenpGV3851::pLGV23neo-transformed tobacco tissue; lane 6, intactchloroplasts from green pGV3851::pLGV23neo-transformed tobacco tissue;lane 7, crude extract from green pGV3851::pGSSTneo3-transformed tobaccotissue; lane 8, intact chloroplasts from greenpGV3851::pGSSTneo3-transformed tobacco tissue. P.K. (?): non-specificband present in untransformed plant tissue, probably due to the activityof plant kinase.

Methods: Three grams of green callus were homogenised by a few shortbursts at low speed in a Waring Blendor in GR buffer (0.33 M sorbitol,50 mM Hepes-KOH (pH 7.5), 1 mM MgCl₂ ; 1 mM MnCl2; 1 mM MnCl₂ ; 1 mM Na₂-EDTA, 2 mM Na₂ -EDTA, 1 mg/ml isoacorbate, 0.5 mg/ml BSA). Thehomogenate was filtered through two layers of Miracloth and the filtratewas centrifuged from 0 to 4340×g and braked in the shortest possibletime. The crude chloroplasts pellet was resuspended in a few ml of GRbuffer. Intact chloroplasts were prepared from crude chloroplastspellets by sedimentation in Percoll density gradients⁵³.Gradient-purified intact chloroplasts were washed with GR and lysed in25 mM Tris-HCl (pH 7.5) containing 0.5% β-mercapto ethanol.

Crude callus extracts were prepared by homogenizing 70 mg tissue in 70μl extraction buffer (1% β-mercaptoethanol; 50 mM Tris; pH 6.8; 0.13mg/ml leu-peptine) and clearing of the homogenate (2 minutes at 18,800g). Crude extracts of E. coli were prepared by sonication in a buffercontaining 10 mM Tris.HCl (pH 7.5); 10 mM MgCl₂ ; 25 mM NH₄ Cl and 10 mMDTT^(ref).60, followed by centrifugation to remove cellular debris. Theassay for NPT(II) activity is a modification of the in situ detectionmethod described⁴⁷. Samples diluted with a 10× loading buffer (50%glycerol; 0.5% SDS; 10% β-mercaptoethanol; 0.005% bromophenol blue) wereseparated on a 10% (w/v) nondenaturing polyacrylamide gel. Afterelectrophoresis, the gel was washed twice for 10 minutes with distilledwater and equilibrated for 30 minutes in 2× reaction buffer (100 mMTris, pH 7.5; 50 mM MgCl₂ ; 400 mM NH₄ Cl; 1 mM DTT). The gel was thentransferred onto a glass plate and overlaid with a 1% agarose gelcontaining 30 μg/ml kanamycin sulphate and 200 μCi γ³² P-ATP in 1×reaction buffer. After 30 minutes at room temperature, the gel sandwichwas covered with Whatman P81 phosphocellulose paper, two sheets ofWhatman 3MM paper, and a stack of blotting paper pressed by weight (1kg) to allow binding of the phosphorylated kanamycin to the P81 paper ina Southern-type transfer. After 3 hours the P81 paper was washed for 5minutes with 500 ml hot water (80° C.), and for 3 hours several timeswith a 50 mM sodium phosphate buffer (pH 7.0). The p81 paper was driedand autoradiographed overnight using an intensifying screen to visualisethe radio-labelled kanamycin formed at the position where the proteinswith NPT(II) activity migrate in the polyacrylamide gel.

6) Localization of NPT(II) activity in the stromal fraction ofchloroplasts isolated from pGV3851::pGSSTneo3-transformed tobacco tissue(FIG. 11)

Intact chloroplasts were isolated from pGV3851::pGSSTneo3-transformedcallus tissue and fractionated into stromal and membrane fractions. TheNPT(II) activity associated with each of these fractions was assayed.Lane 1, E. coli extract containing NPT(II); lane 2, E. coli extractcontaining TP-NPT(II); lane 3, stromal fraction of chloroplasts isolatedfrom green pGV3851::pGGSSTneo3-transformed tobacco tissue; lane 4,membrane fraction of chloroplasts in lane 3; lane 5, was of the membranefraction shown in lane 4. P. K. (?), see FIG. 10.

Intact chloroplasts were isolated from greened tobacco tissue asdescribed in the legend to FIG. 10. Chloroplasts washed twice withsorbitol-Hepes buffer and recovered by centrifugation were fractionatedintro stroma and membrane portions by resuspending plastids in 25 mMTris-HCl (pH 7.5) containing 0.5% β-mercaptoethanol followed bycentrifugation at 18,800× g. Membrane fractions were twice washed andpelleted to remove residual stromal contamination. Wash fractions wereroutinely tested for residual NPT(II) activity.

7) Protection of the NPT(II) activity present within chloroplasts ofpGV3851::pGSST-neo3-transformed tobacco cells to protease treatment(FIG. 12)

Intact chloroplasts isolated from pGV3851::pGSSTneo3-transformed tobaccocallus tissue were subjected to limited proteolytic digestion and thenfractionated into the stromal and membrane components. The proteaseinsensitivity of NPT(II) activity associated with these fractions wasassayed. Lane 1, E. coli extract containing NPT(II); lane 2, stromalfraction of intact chloroplasts isolated from greenpGV3851::pGSSTneo3-transformed tobacco tissue and lysed before proteasetreatment; lane 3, stromal fraction on non-protease-treated intactchloroplasts isolated from green PGV3851::pGSSTneo3-transformed tobaccotissue; lane 4, stromal fraction of protease-treated intact chloroplastsisolated from green pGV3851:: pGSSTneo3-transformed tobacco tissue; P.K. (?), see FIG. 3.

Intact chloroplasts were prepared from greened tobacco callus tissue asdescribed in the legend to FIG. 10. Protease treatment of isolatedchloroplast was carried out as previously described⁵³. Protease-treatedand untreated plastids were fractionated as described in the legend toFIG. 5.

8) In vitro uptake of TP-NPT(II) fusion protein by isolated peachloroplasts (FIG. 13)

An autoradiogram showing the in situ localization of NPT(II) activity inbacterial and chloroplast fractions following fractionation onnon-denaturing polyacrylamide gels is presented. Lane 1, extract from E.coli harbouring pBR322::Tn5 (NPT(II)); lane 2, extract from E. coliharbouring pGLTneo1 (TP-NPT(II)); lane 3, stromal fraction of peachloroplasts prior to incubation with bacterial extracts; lane 4,stromal fraction of pea chloroplasts incubated with bacterial extractscontaining the TP-NPT(II) fusion protein; lane 5, stromal fractions ofprotease-treated pea chloroplasts (same amount as in lane 4) incubatedwith bacterial extracts containing the TP-NPT(II) fusion protein; lane6, washed membrane fractions of the same chloroplasts as in lane 5; lane7, washed membrane fraction of the same chloroplasts as in lane 4.

Methods

Intact chloroplasts were isolated from pea (Pisum sativum) leaves bysedimentation through Percoll density gradients⁵³. Intact chloroplastswere washed and resuspended in sorbitol-Hepes buffer (50 mM Hepes-KOH,pH 7.5; 0.33 M sorbitol) and stored at 0° C. In vitro uptake intoisolated chloroplasts was carried out essentially as described⁵³ exceptthat the incubation mix was modified for use with bacterial extracts.Uptake reactions (300 μl final volume) contained intact chloroplasts(equivalent to 200-300 μg chlorophyll) and 50 μl of bacterial extract(as described in the legend to FIG. 10) in buffer containing 0.33 Msorbitol, 50 mM Hepes.KOH (pH 7.5), 1 mM MgCl₂, 1 mM Na₂ -EDTA.Following incubation at 20-22° C. in the light with gentle shaking for 1hour, chloroplasts were diluted with sorbitol-Hepes buffer and intactchloroplasts recovered by centrifugation at 4340× g. Chloroplasts washedtwice with sorbitol-Hepes buffer and recovered by centrifugation wereeither fractioned immediately (see legend to FIG. 11) or subjected toprotease treatment as previously described⁵³. Aliquots of samples wereeither assayed immediately for NPT(II), or stored at -80° C. and assayedat a later time.

The results presented in this example from both the in vivo and in vitrostudies clearly demonstrate that the NPT(II) component of the TP-NPT(II)fusion protein is translocated across the chloroplast envelope and isfinally located in the stroma. The requirement of the transit peptidefor this process is shown by the failure to detect uptake of NPT(II) bychloroplasts, when the transit peptide has not been fused to NPT(II).The TP-NPT(II) fusion protein, however, bears no similarity in theaminoacid sequence to the small subunit precursor, particularly near tothe processing site thereof immediately following the transit peptide.This suggests that all of the sequence information required fortranslocation resides within the transit peptide.

Under normal physiological growth conditions for plants, the smallsubunit precursor is rapidly taken up and processed by the chloroplasts,and a large free pool of unprocessed precursor is not observed¹, 10. Ithas been shown here, that in tobacco cells transformed withpGV3851::pGSSTneo3, all of the NPT(II) activity observed in either crudecellular extracts or isolated chloroplast fractions migrates on the gelsystem⁴⁷ used with similar electrophoretic mobility to the originalNPT(II). Processing of the TP-NPT(II) fusion protein is presumablycarried out by the same soluble, chloroplast-associated protease¹⁸ thatis responsible for the processing of the small subunit precursor. Itseems likely, therefore, that the processing of the TP-NPT(II) fusionprotein occurs at the same Cys/Met site (FIG. 8B) used in the smallsubunit precursor. Thus it can be hypothesized that the transit peptidecan mediate not only translocation, but also site-specific processing.Furthermore, both the translocation and processing steps apparentlyoccur rather efficiently in pGV3851::pGSSTneo3-transformed tobaccocells, since within the detection limits of our assay system, all of theNPT(II) activity observed corresponds to the processed form of theTP-NPT(II) fusion protein.

The results presented here again clearly demonstrate the applicabilityof using Agrobacterium-mediated cell transformation to introduce foreigngenes into plants.

EXAMPLE III

Construction of a plasmid encoding a chimaeric gene encoding theTP-NPT(II) fusion protein and wherein the coding sequences are under thecontrol of a foreign promotor (FIG. 14)

The construction starts from pGSST neo3. This plasmid was then digestedwith EcoRI and Hind III. The staggered ends of the long fragments werefilled in with the Klenow polymerase. The DNA obtained was then ligatedto a SauIIIA fragment (270 bp) originating from plasmid pLGV 2382.

This SauIIIA fragment contains the promoter of the nopaline synthase(HERRERA-ESTRELLA L. et al (1983) Embo. J., 2, 987-995). The latterfragment was also treated with the klenow fragment of the DNApolymerase. The SauIIIA fragment so repaired and the repaired fragmentfrom pGSSTneo3 were then ligated with T4 ligase, whereby plasmidpLSSTneo1 was obtained. The plasmid containing the promoter regionoriented in the proper direction was identified by restriction analysiswith the SacII restriction enzyme and BamHI. The plasmid (pLSSTneo1)which proved to contain the biggest SacII-BamHI fragment was also theone which contained the promoter region and the TP-NPT II fragment inthe proper orientation and under control of said promoter.

There is thus, shown another plasmid having this time a constitutivepromoter instead of the normal leaf specific light-inducible promoter.Consequently, a plasmid was obtained which can cause the protein locateddownstream of the promoter to be expressed also in the dark and also inother tissues of the plant. In such a manner one controls the level ofproduction of metabolites of interest, for instance fatty acids or aminoacids.

It will be appreciated that the invention also makes it possible to puta gene normally expressed under photosynthetic conditions under thecontrol of a promoter which is normally operative in a constant manner(day and night). In such a way and for instance one can obtain theconstant production of a determined amino acid under the control of apromoter operative in seeds.

The invention thus opens the way to important agricultural applicationsinvolving chloroplast functions. More particularly it enables theintroduction of proteins of controlled structure in plant-cellschloroplast. These proteins can be introduced into the chloroplasteither as such or as fusions with proteins or protein subunits which arecoded for by natural genes and normally transported into the plant cellchloroplast. These proteins may either be proteins foreign to the plantcells to be transformed or be similar to endogenous proteins, yetdifferent there from by controlled mutations. Particularly the inventionnow provides for the possibility of modifying at will genes including adetermined protein, for instance for the sake of improving the activityof the enzyme encoded by the chloroplast genes. The invention alsoprovides for the possibility of substituting another promoter for theendogenous promoter included in the natural gene to thereby regulate ina controlled manner the production of the chloroplast proteins.

The invention further provides valuable tools for a better understandingof the role played by various domains of transported proteinsinteracting with chloroplast coded proteins. It also renders possiblethe study of whether determined chimaeric genes can direct the transferof proteins normally encoded by the chloroplast back into thisorganelle. Model systems of chloroplast-encoded genes of importance forbasic research and agricultural application are readily available, suchas the large subunit of RuBP carboxylase, which contains the catalyticsite of the holoenzyme, or the 32 K protein conferring resistance tocertain herbicides. The similarity between the results obtained from invivo and in vitro studies also suggests that the production in E. coliof fusion proteins composed of segments of nuclear-encoded organellepolypeptides and an enzymatic reporter is a powerful technique for therapid analysis of the signals and processes involved in protein importby isolated organelles.

The invention further provides the means which enables chimaericengineering of plants with a potential for amino acid overproduction orimprovement of plant productivity, and therefore meets needs which havealready been recalled in the preamble of this application.

The use of transit peptides for specifically targeting polypeptides inthe chloroplast also provides the possibility of genetically engineeringgenes containing sequences encoding key enzymes of given pathways insuch manner that said key enzymes are no longer subjected to the normalregulation systems included in the natural plant cells.

The invention also provides means for solving other problems that havebeen mentioned in the preamble i.e. the production of herbicideresistant plants. Actually the invention now provides a method forfusion of a "second sequence" encoding the protein of interest with afirst sequence encoding a transit peptide, the chimaeric gene soproduced being capable after its insertion in the genetic DNA of thecells of the plant of interest to control the translocation of theprotein of interest into the chloroplasts.

The invention opens the way to many other applications. A few additionalexamples are illustrated hereafter and in which the enzyme Ribulosebisphosphate carboxylase (RuBPCase) can be brought into play.

a) Improvement of the carboxylase/oxidase ratio

This enzyme catalyzes two enzymatic reactions:

1) The condensation of a molecule of Ribulose bisphosphate with amolecule of CO₂ to form two molecules of phosphoglyceric acid(Carboxylase reaction).

2) Reaction of a ribulose bisphosphate molecule with a molecule ofoxygen to produce phosphoglycolate (oxidase reaction).

The latter is a competitive reaction with the carboxylation. Thereforeit limits the efficiency of conversion of CO₂ into organic compounds.

The invention now provides a technique of site directed mutagenesiswhich allows the controlled alteration of a determined protein to begiven full effect. For instance the modification of the RuBPCase in sucha way that the carboxylase/oxidase ratio is much more favourable can nowbe contemplated. Another approach is to simply take a gene encoding forthe RuBPCase from another plant or from another organism such ascyanobacteria which have a more favourable ratio, to fuse it with anucleic acid fragment containing a promoter and a transit peptideeffective in the plant of interest and to introduce the chimaeric geneobtained into said plant.

b) Improvement of plant productivity

There are several factors limiting plant productivity such as lack ofnutrients and a low efficiency in light harvesting or CO₂ assimilation.Since the lack of nutrients can be solved using fertilizers, one of themain limiting factor for plant productivity becomes CO₂ assimilation.CO₂ uptake by a leaf depends mostly on two factors:

1) The physical diffusion of CO₂ along the plant cells and

2) the efficiency of CO₂ conversion to organic compounds.

Although different pathways for CO₂ assimilation exist in higher plants,they share the same limiting step, which is the efficiency of theRuBPCase enzyme. Here again the invention provides means for overcomingthis problem at least in part, for instance upon introducing in thecells of the plant a chimaeric gene comprising sequences fused with oneanother and which respectively contain a promoter region and a fragmentencoding a transit peptide which are particularly effective in thatplant, on the one hand, and a sequence encoding a more efficientRuBPCase and originating from another plant, on the other hand.

Cultures comprising plasmids, intermediate cloning vectors, andmicroorganisms prepared by the processes of this invention areexemplified by cultures deposited in the German Collection ofMicroorganisms (DSM), Guttingen, Germany. These cultures are identifiedhereafter:

(1) E. coli HB101 (pSRP6)

(2) E. coli HB101 (pKM 109/9)

(3) E. coli HB101 (pGSST3)

These cultures were deposited on Dec. 27, 1984.

These cultures were assigned accession numbers 3172 (1) 3171 (2) 3170(3).

Other cultures referred to in this application have also been depositedon or before December 27th, i.e. on Dec. 20th, 1984. Plasmids weremaintained in the microorganisms identified in the left hand part of thetable hereafter.

These cultures have been assigned the following accessing numbers:

    ______________________________________                                        Internal Code/  Plasmid                                                         Taxonomic Designation in strain DSM No.                                     ______________________________________                                        AZ 1/           p PSR 6 delta R V                                                                          3161                                               E. coli K12 and VII                                                           AZ 2/ p I-22 3162                                                             E. coli K12 and VII                                                           AZ 3/ p II-4 3163                                                             E. coli and VII                                                               AZ 4/ pGV 710 3164                                                            E. coli K12 and VII                                                           AZ 5/ pGV 3850::pSNIPP 3165                                                   AGR. TUMEF. VII                                                               AZ 6/ pGV 3850::pSNIF 3166                                                    AGR. TUMEF.                                                                   AZ 7/ pGV 3850 3167                                                           AGR. TUMEF. VII                                                             ______________________________________                                    

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Herrera-Estrella, L., Depicker, A., Van Montagu, M., and Schell, J.(1983), Nature 303, 209213.

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    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 6                                           - -  - - (2) INFORMATION FOR SEQ ID NO: 1:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 171 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Pisum sat - #ivum                                      - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION:1..171                                                           (D) OTHER INFORMATION:/not - #e= "transit peptide associated                      with the - #small subunit of RuBP of pea c..."                  - -    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                               - - ATGGCTTCTA TGATATCCTC TTCCGCTGTG ACAACAGTCA GCCGTGCYTC TA -             #KGGGGCAA     60                                                                 - - TCCGCSGCAG TGGCTCCATT CGGCGGCCTS AAATCCATGA CTGGATTCCC AG -            #TGAAGAAG    120                                                                 - - GTCAACACTG ACATTACTTC CATTACAAGC AATGGTGGAA GAGTAAAGTG C - #                171                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO: 2:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 57 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Pisum sat - #ivum                                      - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION:1..57                                                            (D) OTHER INFORMATION:/not - #e= "transit peptide of the                           small sub - #unit of RuBP of pea cells."                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #2:                           - - Met Ala Ser Met Ile Ser Ser Ser Ala Val Th - #r Thr Val Ser Arg Ala      1               5   - #                10  - #                15               - - Ser Arg Gly Gln Ser Ala Ala Val Ala Pro Ph - #e Gly Gly Leu Lys Ser                  20      - #            25      - #            30                   - - Met Thr Gly Phe Pro Val Lys Lys Val Asn Th - #r Asp Ile Thr Ser Ile              35          - #        40          - #        45                       - - Thr Ser Asn Gly Gly Arg Val Lys Cys                                          50              - #    55                                                  - -  - - (2) INFORMATION FOR SEQ ID NO: 3:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 108 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION:1..108                                                           (D) OTHER INFORMATION:/not - #e= "transit peptide  of the                          chlorophyll - #a/b-protein complex"                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #3:                           - - ATGGCCGCAT CATCATCATC ATCCATGGCT CTCTCTTCTC CAACCTTGGC TG -             #GCAAGCAA     60                                                                 - - CTCAAGCTGA ACCCATCAAG CCAAGAATTG GGAGCTGCAA GGTTCACC  - #                   108                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO: 4:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION:1..36                                                            (D) OTHER INFORMATION:/not - #e= "transit peptide of the                           light har - #vesting chlorophyll a/b protein complex"           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #4:                           - - Met Ala Ala Ser Ser Ser Ser Ser Met Ala Le - #u Ser Ser Pro Thr Leu      1               5   - #                10  - #                15               - - Ala Gly Lys Gln Leu Lys Leu Asn Pro Ser Se - #r Gln Glu Leu Gly Ala                  20      - #            25      - #            30                   - - Ala Arg Phe Thr                                                                  35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 5:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Pisum sat - #ivum                                      - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION:1..18                                                            (D) OTHER INFORMATION:/not - #e= "region around the                                processing - #site of the precursor to the small subunit                      polypeptide - #of Rubisco encoded by the pea ss3.6 gene"        - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Cleavage-sit - #e                                               (B) LOCATION:9                                                       - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #5:                           - - Ser Asn Gly Gly Arg Val Lys Cys Met Gln Va - #l Trp Pro Pro Ile Gly      1               5   - #                10  - #                15               - - Lys Lys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 6:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: chimeric                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION:1..18                                                            (D) OTHER INFORMATION:/not - #e= "fusion region of the transit                     peptide f - #rom Rubisco small subunit to the NPTII           protein"                                                                         - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Cleavage-sit - #e                                               (B) LOCATION:9                                                       - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION:17..18                                                           (D) OTHER INFORMATION:/not - #e= "amino acid residues derived                     from the - #original NPTII protein"                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #6:                           - - Ser Asn Gly Gly Arg Val Lys Cys Met Asp Pr - #o Ala Asn Leu Ala Trp      1               5   - #                10  - #                15               - - Ile Glu                                                                 __________________________________________________________________________

We claim:
 1. A plant comprising in its nuclear genome a chimaeric DNAsequence, comprising:a.) a nucleic acid sequence coding for a chimaericprotein or polypeptide comprising:i.) a transit peptide of a cytoplasmicprecursor of a chloroplast protein or chloroplast polypeptide of a plantspecies; and ii.) a protein or polypeptide of interest which isdifferent from the polypeptide or protein normally associated with saidtransit peptide in a cytoplasmic precursor, wherein said transit peptideis located in the amino-terminal part of said chimaeric protein orpolypeptide; and b.) a promoter upstream of said nucleic acid sequencerecognized by polymerases endogenous to said plant; wherein thechimaeric DNA sequence can be expressed in cells of said plant undercontrol of said promoter, and said protein or polypeptide of interest istranslocated into chloroplasts of cells of said plant.
 2. The plant ofclaim 1, in which said protein or polypeptide of interest confersresistance to an herbicide.
 3. The plant of claim 2, in which saidprotein or polypeptide of interest is an acetolactate synthase.
 4. Theplant of claim 2, in which said protein or polypeptide of interest is5-enol pyruvyl-3-phosphoshikimate synthase.
 5. The plant of claim 1, inwhich said protein or polypeptide of interest is of bacterial origin. 6.The plant of claim 1, in which said protein or polypeptide of interestis of plant origin.
 7. The plant of claim 1, in which the first aminoacid of said protein or polypeptide of interest is a methionine.
 8. Theplant of claim 1, in which no more than the first seven amino acids ofsaid protein or polypeptide of interest are encoded by a syntheticnucleotide linker.
 9. The plant of claim 1, in which said transitpeptide is from a cytoplasmic precursor of a chloroplast proteinselected from the group consisting of the small subunit ofribulose-1,5-bisphosphate carboxylase, and chlorophyll a/b bindingprotein.
 10. The plant of claim 9, in which the transit peptide is froma cytoplasmic precursor of the small subunit ofribulose-1,5-bisphosphate carboxylase of a plant selected from the groupconsisting of soybean, pea, duckweed, and wheat.
 11. The plant of claim9 in which the transit peptide has the sequence selected from the groupconsisting of the amino acid sequence of SEQ ID No 2 and the amino acidsequence of SEQ ID No
 4. 12. The plant of claim 1, in which saidpromoter is normally associated with the DNA encoding said transitpeptide.
 13. The plant of claim 12, in which said promoter is a promoterof a gene selected from the group consisting of the plastocyanin gene,the ferredoxin- NADP+oxydoreductase gene, the ribulose-1,5-bisphosphatecarboxylase gene, and the chlorophyll a/b binding protein gene.
 14. Theplant of claim 1, in which said promoter is foreign to the DNA encodingsaid transit peptide.
 15. The plant of claim 14, in which said promoteris a promoter of the nopaline synthase gene.
 16. A plant comprising inits nuclear genome a chimaeric DNA sequence comprising:a.) a nucleicacid sequence coding for a chimaeric protein or polypeptide comprisingin sequence:(1) a transit peptide of a cytoplasmic precursor of achloroplast protein or chloroplast polypeptide of a plant species, and(2) a hybrid protein comprising in sequence:(i) a linker, and (ii) aprotein or polypeptide of interest which is different from thepolypeptide or protein normally associated with said transit peptide insaid cytoplasmic precursor wherein said linker does not significantlyalter the biological property of said protein or polypeptide of interestin said hybrid protein, and b.) a promoter upstream of said nucleic acidsequence recognized by polymerases endogenous to a plant, wherein thechimaeric DNA sequence is expressed in cells of said plant under thecontrol of said promoter, and said hybrid protein is translocated intothe chloroplasts of said cells of the plant.
 17. The plant of claim 16,wherein said linker is a N-terminal part of a chloroplast protein orchloroplast polypeptide derived from a natural cytoplasmic precursorthereof by proteotypical removal of its transit peptide.
 18. The plantof claim 16, in which said protein or polypeptide of interest is ofbacterial origin.
 19. The plant of claim 16, in which said protein orpolypeptide of interest is of plant origin.
 20. The plant of claim 16,in which said protein or polypeptide of interest confers resistance toan herbicide.
 21. The plant of claim 16, in which said protein orpolypeptide of interest is an acetolactate synthase.
 22. The plant ofclaim 16, in which said protein or polypeptide of interest is 5-enolpyruvyl-3-phosphoshikimate synthase.
 23. The plant of claim 16, in whichsaid N-terminal part is encoded by a DNA sequence that comprises anintron.
 24. The plant of claim 16, in which said transit peptide is froma cytoplasmic precursor of a chloroplast protein selected from the groupconsisting of the small subunit of ribulose-1,5-bisphosphatecarboxylase, and chlorophyll a/b binding protein.
 25. The plant of claim24, in which the transit peptide is from a cytoplasmic precursor of thesmall subunit of ribulose-1,5-bisphosphate carboxylase of a plantselected from the group consisting of soybean, pea, duckweed and wheat.26. The plant of claim 24, in which the transit peptide has the sequenceselected from the group consisting of the amino acid sequence of SEQ IDNo 2 and the amino acid sequence of SEQ ID No.
 4. 27. The plant of claim16, in which said promoter is normally associated with the DNA encodingsaid transit peptide.
 28. The plant of claim 27, in which said promoteris a promoter of a gene selected from the group consisting of theplastocyanin gene, the ferredoxin-NADP+oxydoreductase gene, theribulose-1,5-bisphosphate carboxylase gene, and the chlorophyll a/bbinding protein gene.
 29. The plant of claim 16, in which said promoteris foreign to the DNA encoding said transit peptide.
 30. The plant ofclaim 29, in which said promoter is a promoter of the nopaline synthasegene.
 31. A plant comprising in its genome a chimaeric DNA sequencecomprising:a.) a nucleic acid sequence coding for a chimaeric protein orpolypeptide comprising in sequence:(1) a transit peptide of acytoplasmic precursor of a chloroplast protein or chloroplastpolypeptide of a plant species, and, (2) a fusion protein comprising insequence,(i) a N-terminal part consisting of no more than the first 22N-terminal amino acids of the amino acid sequence of a cytoplasmicprecursor of a chloroplast protein or polypeptide after prototypicalremoval of its transit peptide upon translocation into the chloroplastof a cell of a plant, (ii) a protein or polypeptide of interest which isdifferent from the polypeptide or protein normally associated with saidtransit peptide, and, b.) a promoter upstream of said nucleic acidsequence recognized by polymerases endogenous to said plant, wherein thechimaeric DNA sequence is expressed in cells of said plant under thecontrol of said promoter, and said fusion protein is translocated intothe chloroplasts of said cells of said plants.
 32. An image formingaparatus on which at least one set of ink jet recording heads ismountable, each of the ink jet recording heads of a same set dischargingone of a plurality of inks which belong to a same color family and havedifferent densities, ink jet recording heads of a same set comprise athick ink jet recording head associated with thick ink of one colorfamily, and a thin ink jet recording head associated with thin ink ofthe one color family, the image forming apparatus comprising:detectingmeans for detecting a failure of ink discharge of the ink jet recordingheads; mode selecting means for selecting, in response to a detectionsignal outputted from said detecting means, a recording mode of the inkjet recording heads from among i) a mode to use both of thick and thinink, ii) a mode to use only thin ink, and iii) a mode to use only thickink; first control means for controlling the driving of the ink jetrecording heads in accordance with a recording mode selected by saidmode selecting means; and second control means for setting a recordingdensity on a recording medium in accordance with the mode selected bythe mode selection means, wherein said mode selecting means selects themode which uses only thick ink from the thick ink head of each set ifthe detection signal indicates that ink discharge of the thin ink headhas failed, and the mode which uses only thin ink from the thin ink headof each set if the detection signal indicates that ink discharge of thethick ink head has failed.
 33. The plant of claim 31, in which saidprotein or polypeptide of interest is of plant origin.
 34. The plant ofclaim 31, in which said protein or polypeptide of interest confersresistance to an herbicide.
 35. The plant of claim 34, in which saidprotein or polypeptide of interest is an acetolactate synthase.
 36. Theplant of claim 34, in which said protein or polypeptide of interest is5-enol pyruvyl-3-phosphoshikimate synthase.
 37. The plant of claim 31,in which said N-terminal part is encoded by a DNA sequence thatcomprises an intron.
 38. The plant of claim 31, in which said N-terminalpart is from the small subunit of the ribulose-1,5-bisphosphatecarboxylase of Pisum sativum.
 39. The plant of claim 31, in which saidN-terminal part consists of no more than the first five N-terminal aminoacids of said amino acid sequence of a cytoplasmic precursor of achloroplast protein or polypeptide after proteotypical removal of itstransit peptide upon translocation into the choroplast of a cell of aplant.
 40. The plant of claim 39, in which said N-terminal part has thesequence M-Q-V-W-P.
 41. The plant of claim 31, in which said transitpeptide is from a cytoplasmic precursor of a chloroplast proteinselected from the group consisting of the small subunit ofribulose-1,5-bisphosphate carboxylase, and chlorophyll a/b bindingprotein.
 42. The plant of claim 31, in which the transit peptide is froma cytoplasmic precursor of the small subunit ofribulose-1,5-bisphosphate carboxylase of a plant selected from the groupconsisting of soybean, pea, duckweed, and wheat.
 43. The plant of claim31 in which the transit peptide has the sequence selected for the groupconsisting of the amino acid sequence of SEQ ID No 2 and the amino acidsequence of SEQ ID No.
 4. 44. The plant of claim 31, in which saidpromoter is normally associated with the DNA encoding said transitpeptide.
 45. The plant of claim 44, in which said promoter is a promoterof a gene selected from the group consisting of the plastocyanin gene,the ferredoxin-NADP+oxydoreductase gene, the ribulose-1,5-bisphosphatecarboxylase gene, and the chlorphyll a/b binding protein gene.
 46. Theplant of claim 31, in which said promoter is foreign to the DNA encodingsaid transit peptide.
 47. The plant of claim 46, in which said promoteris a promoter of the nopaline synthase gene.
 48. A seed of the plant ofany one of claims 1 to 6, 9, 16, 17, and 31, comprising said chimaericDNA sequence.
 49. A cell of the plant of any one of claims 1 to 6, 9,16, 17, and 31, comprising said chimaeric DNA sequence.